Human G protein-coupled receptors and modulators thereof for the treatment of metabolic-related disorders

ABSTRACT

The present invention relates to methods of identifying whether a candidate compound is a modulator of a G protein-coupled receptor (GPCR). In preferred embodiments, the GPCR is human. In other preferred embodiments, the GPCR is coupled to Gi and lowers the level of intracellular cAMP. In other preferred embodiments, the GPCR is expressed endogenously by adipocytes. In further preferred embodiments, the GPCR inhibits intracellular lipolysis. In other further preferred embodiments, the GPCR is a nicotinic acid receptor. The present invention also relates to methods of using a modulator of said GPCR. Preferred modulator is agonist. Agonists of the invention are useful as therapeutic agents for the prevention or treatment of metabolic-related disorders, including dyslipidemia, atherosclerosis, coronary heart disease, stroke, insulin resistance, and type 2 diabetes.

The present application is a Continuation of U.S. Utility patentapplication Ser. No. 10/897,815, filed Jul. 23, 2004, which is aContinuation of U.S. Utility patent application Ser. No. 10/314,048,filed Dec. 6, 2002, which is a Continuation-In-Part of U.S. Utilitypatent application Ser. No. 10/096,511, filed Mar. 12, 2002, (nowabandoned), which is a Continuation of U.S. Utility patent applicationSer. No. 09/995,543, filed Nov. 27, 2001 (now abandoned) and claimsbenefit of priority to U.S. Provisional Patent Applications: Ser. No.60/399,917, filed Jul. 29, 2002, Ser. No. 60/404,761, filed Aug. 19,2002 and Ser. No. 60/410,747, filed Sep. 13, 2002, the disclosure ofeach of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to methods of identifying whether acandidate compound is a modulator of a G protein-coupled receptor(GPCR). In preferred embodiments, the GPCR is human. In other preferredembodiments, the GPCR is coupled to Gi and lowers the level ofintracellular cAMP. In other preferred embodiments, the GPCR isexpressed endogenously by adipocytes. In further preferred embodiments,the GPCR inhibits intracellular lipolysis. In other further preferredembodiments, the GPCR is a nicotinic acid receptor. The presentinvention also relates to methods of using a modulator of said GPCR.Preferred modulator is agonist. Agonists of the invention are useful astherapeutic agents for the prevention or treatment of metabolic-relateddisorders, including dyslipidemia, atherosclerosis, coronary heartdisease, stroke, insulin resistance and type 2 diabetes.

BACKGROUND OF THE INVENTION

A. Nicotinic Acid as an Antilipolytic Agent

Atherosclerosis and stroke are the numbers one and number three leadingcauses of death of both men and women in the United States. [See, e.g.,Nature Medicine, Special Focus on Atherosclerosis, (2002) 8:1209-1262;the disclosure of which is hereby incorporated by reference in itsentirely.] Type 2 diabetes is a public health problem that is serious,widespread and increasing [Brownlee M, Nature (2001) 414:813-20 andreferences therein; Zimmet P et al., Nature (2001) 414:782-7 andreferences therein; Saltiel A R et al., Nature (2001) 414:799-806 andreferences therein; the disclosure of each of which is herebyincorporated by reference in its entirety]. Elevated levels of lowdensity lipoprotein (LDL) cholesterol or low levels of high densitylipoprotein (HDL) cholesterol are, independently, risk factors foratherosclerosis and associated cardiovascular pathologies. In addition,high levels of plasma free fatty acids are associated with insulinresistance and type 2 diabetes. One strategy for decreasingLDL-cholesterol, increasing HDL-cholesterol, and decreasing plasma freefatty acids is to inhibit lipolysis in adipose tissue. This approachinvolves regulation of hormone sensitive lipase, which is therate-limiting enzyme in lipolysis. Lipolytic agents increase cellularlevels of cAMP, which leads to activation of hormone sensitive lipasewithin adipocytes. Agents that lower intracellular cAMP levels, bycontrast, would be antilipolytic.

It is also worth noting in passing that an increase in cellular levelsof cAMP down-regulates the secretion of adiponectin from adipocytes[Delporte, M L et al. Biochem J (2002) 367:677-85; the disclosure ofwhich is incorporated by reference in its entirety]. Reduced levels ofplasma adiponectin have been associated with metabolic-relateddisorders, including atherosclerosis, coronary heart disease, stroke,insulin resistance and type 2 diabetes [Matsuda, M et al. J Biol Chem(2002) 277:37487-91 and reviewed therein; the disclosure of which ishereby incorporated by reference in its entirety]. [Also see: Yamauchi Tet al., Nat Med (2002) 8:1288-95; and Tomas E et al., Proc Natl Acad SciUSA (2002) Nov. 27; the disclosure of each of which is herebyincorporated by reference in its entirety.] Globular adiponectinprotected ob/ob mice from diabetes and apoE deficient mice fromatherosclerosis [Yamauchi, T et al. J Biol Chem (2002) November; thedisclosure of which is hereby incorporated by reference in itsentirety]. [Also see Okamoto, Y et al. Circulation (2002) 26:2767-70;the disclosure of which is hereby incorporated by reference in itsentirety.] There is evidence that the regulation of human serumadiponectin levels through modulation of adipocyte intracellular cAMPlevel is independent of adipocyte lipolysis [Staiger H et al., HormMetab Res (2002) 34:601-3; the disclosure of which is herebyincorporated by reference in its entirety].

Nicotinic acid (niacin, pyridine-3-carboxylic acid) is a water-solublevitamin required by the human body for health, growth and reproduction;a part of the Vitamin B complex. Nicotinic acid is also one of theoldest used drugs for the treatment of dyslipidemia. It is a valuabledrug in that it favorably affects virtually all of the lipid parameterslisted above [Goodman and Gilman's Pharmacological Basis ofTherapeutics, editors Harmon J G and Limbird L E, Chapter 36, Mahley R Wand Bersot T P (2001) pages 971-1002]. The benefits of nicotinic acid inthe treatment or prevention of atherosclerotic cardiovascular diseasehave been documented in six major clinical trials [Guyton J R (1998) AmJ Cardiol 82:18U-23U]. Structure and synthesis of analogs or derivativesof nicotinic acid are discussed throughout the Merck Index, AnEncyclopedia of Chemicals, Drugs, and Biologicals, Tenth Edition (1983),which is incorporated herein by reference in its entirety.

Nicotinic acid and currently existing analogs thereof inhibit theproduction and release of free fatty acids from adipose tissue, likelyvia an inhibition of adenylyl cyclase, a decrease in intracellular cAMPlevels, and a concomitant decrease in hormone sensitive lipase activity.Agonists that down-regulate hormone sensitive lipase activity leading toa decrease in plasma free fatty acid levels are likely to havetherapeutic value. The consequence of decreasing plasma free fatty acidsis two-fold. First, it will ultimately lower LDL-cholesterol and raiseHDL-cholesterol levels, independent risk factors, thereby reducing therisk of mortality due to cardiovascular incidence subsequent to atheromaformation. Second, it will provide an increase in insulin sensitivity inindividuals with insulin resistance or type 2 diabetes. Unfortunately,the use of nicotinic acid as a therapeutic is partially limited by anumber of associated, adverse side-effects. These include flushing, freefatty acid rebound, and liver toxicity.

Agonists of antilipolytic GPCRs having limited tissue distributionbeyond adipose may be especially valuable in view of the diminishedopportunity for potentially undesirable side-effects.

The rational development of novel, nicotinic acid receptor agonists thathave fewer side-effects is an area of active investigation, but to dateit has been hindered by the inability to molecularly identify thenicotinic acid receptor. Recent work suggests that nicotinic acid mayact through a specific GPCR [Lorenzen A, et al. (2001) MolecularPharmacology 59:349-357 and reviewed therein; the disclosure of which ishereby incorporated by reference in its entirety]. Furthermore, it isimportant to consider that other receptors of the same class may existon the surface of adipocytes and similarly decrease hormone sensitivelipase activity through a reduction in the level of intracellular cAMPbut without the elicitation of adverse effects such as flushing, therebyrepresenting promising novel therapeutic targets.

B. G Protein-Coupled Receptors

Although a number of receptor classes exist in humans, by far the mostabundant and therapeutically relevant is represented by the Gprotein-coupled receptor (GPCR) class. It is estimated that there aresome 30,000-40,000 genes within the human genome, and of these,approximately 2% are estimated to code for GPCRs. Receptors, includingGPCRs, for which the endogenous ligand has been identified, are referredto as “known” receptors, while receptors for which the endogenous ligandhas not been identified are referred to as “orphan” receptors.

GPCRs represent an important area for the development of pharmaceuticalproducts: from approximately 20 of the 100 known GPCRs, approximately60% of all prescription pharmaceuticals have been developed. Forexample, in 1999, of the top 100 brand name prescription drugs, thefollowing drugs interact with GPCRs (the primary diseases and/ordisorders treated related to the drug is indicated in parentheses):Claritin ® (allergies) Prozac ® (depression) Vasotec ® (hypertension)Paxil ® (depression) Zoloft ® (depression) Zyprexa ®(psychotic disorder)Cozaar ® (hypertension) Imitrex ® (migraine) Zantac ® (reflux)Propulsid ® (reflux disease) Risperdal ® (schizophrenia) Serevent ®(asthma) Pepcid ® (reflux) Gaster ® (ulcers) Atrovent ® (bronchospasm)Effexor ® (depression) Depakote ® (epilepsy) Cardura ®(prostaticypertrophy) Allegra ® (allergies) Lupron ® (prostate cancer) Zoladex ®(prostate cancer) Diprivan ® (anesthesia) BuSpar ® (anxiety) Ventolin ®(bronchospasm) Hytrin ® (hypertension) Wellbutrin ® (depression)Zyrtec ® (rhinitis) Plavix ® (MI/stroke) Toprol-XL ® (hypertension)Tenormin ® (angina) Xalatan ® (glaucoma) Singulair ® (asthma) Diovan ®(hypertension) Harnal ® (prostatic hyperplasia) (Med Ad News 1999 Data).

GPCRs share a common structural motif, having seven sequences of between22 to 24 hydrophobic amino acids that form seven alpha helices, each ofwhich spans the membrane (each span is identified by number, i.e.,transmembrane-1 (TM-1), transmembrane-2 (TM-2), etc.). The transmembranehelices are joined by strands of amino acids between transmembrane-2 andtransmembrane-3, transmembrane-4 and transmembrane-5, andtransmembrane-6 and transmembrane-7 on the exterior, or “extracellular”side, of the cell membrane (these are referred to as “extracellular”regions 1, 2 and 3 (EC-1, EC-2 and EC-3), respectively). Thetransmembrane helices are also joined by strands of amino acids betweentransmembrane-1 and transmembrane-2, transmembrane-3 andtransmembrane-4, and transmembrane-5 and transmembrane-6 on theinterior, or “intracellular” side, of the cell membrane (these arereferred to as “intracellular” regions 1, 2 and 3 (IC-1, IC-2 and IC-3),respectively). The “carboxy” (“C”) terminus of the receptor lies in theintracellular space within the cell, and the “amino” (“N”) terminus ofthe receptor lies in the extracellular space outside of the cell.

Generally, when a ligand binds with the receptor (often referred to as“activation” of the receptor), there is a change in the conformation ofthe receptor that facilitates coupling between the intracellular regionand an intracellular “G-protein.” It has been reported that GPCRs are“promiscuous” with respect to G proteins, i.e., that a GPCR can interactwith more than one G protein. See, Kenakin, T., 43 Life Sciences 1095(1988). Although other G proteins exist, currently, Gq, Gs, Gi, Gz andGo are G proteins that have been identified. Ligand-activated GPCRcoupling with the G-protein initiates a signaling cascade process(referred to as “signal transduction”). Under normal conditions, signaltransduction ultimately results in cellular activation or cellularinhibition. Although not wishing to be bound to theory, it is thoughtthat the IC-3 loop as well as the carboxy terminus of the receptorinteract with the G protein.

Gi-coupled GPCRs lower intracellular cAMP levels. The Melanophoretechnology (see infra) is useful for identifying Gi-coupled GPCRs.

Under physiological conditions, GPCRs exist in the cell membrane inequilibrium between two different conformations: an “inactive” state andan “active” state. A receptor in an inactive state is unable to link tothe intracellular signaling transduction pathway to initiate signaltransduction leading to a biological response. Changing the receptorconformation to the active state allows linkage to the transductionpathway (via the G-protein) and produces a biological response.

A receptor may be stabilized in an active state by a ligand or acompound such as a drug. Recent discoveries, including but notexclusively limited to modifications to the amino acid sequence of thereceptor, provide means other than ligands or drugs to promote andstabilize the receptor in the active state conformation. These meanseffectively stabilize the receptor in an active state by simulating theeffect of a ligand binding to the receptor. Stabilization by suchligand-independent means is termed “constitutive receptor activation.”

SUMMARY OF THE INVENTION

RUP25, RUP38, RUP19 and RUP11 belong to a sub-family of human GPCRs onthe basis of homology at the nucleotide level. See, Tables B and C,infra. Polynucleotide sequence and polypeptide sequence for human (h),rat (r), or mouse (m) RUP25, RUP38, RUP19, or RUP11 is provided in theSequence Listing (also see, Tables E and F infra for corresponding SEQ.ID. NOs.).

Agonist engagement of Gi-coupled GPCRs is known to lead to loweredlevels of intracellular cAMP. Lower levels of cAMP in adipocytes lead todiminished hormone sensitive lipase activity. (See, supra.) The presentinvention is based in part on the discovery by Applicant that GPCRsRUP25, RUP38, and RUP19 are coupled to Gi and expressed endogenously byadipocytes. RUP38 and RUP19 are further shown by Applicant to havelimited tissue distribution beyond adipose. RUP11 is also disclosed tobe coupled to Gi.

Applicant discloses herein that RUP25 is a nicotinic acid and anantilipolytic GPCR. Applicant further discloses that RUP38 and RUP19 areantilipolytic GPCRs. RUP11 is also disclosed to be antilipolytic. Thepresent invention is directed in part to methods of identifying whethera candidate compound is a modulator of RUP25, RUP38, RUP19 or RUP11. Thepresent invention also relates to methods of using said modulator ofRUP25, RUP38, RUP19 or RUP11. Preferred said modulator is an agonist.Agonists of RUP25, RUP38, RUP19 or RUP11 are useful as therapeuticagents for the prevention or treatment of metabolic-related disorders,including dyslipidemia, atherosclerosis, coronary heart disease, stroke,insulin resistance, and type 2 diabetes.

Nicotinic acid is disclosed by Applicant to be an agonist for RUP25 butnot for RUP38 or RUP19. (−)-Nicotine is also disclosed to be an agonistfor RUP25. Exposure of cells expressing RUP25 to nicotinic acid is shownby Applicant to lower the level of intracellular cAMP. Exposure ofisolated rat epididymal adipocyte RUP25 to nicotinic acid is shown byApplicant to inhibit lipolysis. Exposure of RUP25 within adipocyteprimary cultures derived from human subcutaneous fat to nicotinic acidis shown by Applicant also to inhibit lipolysis. In vivo administrationof nicotinic acid to rats is shown by Applicant to lower plasma freefatty acids (FFA).

Applicant has identified(5-Hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanone tobe an agonist for RUP25 but not for RUP38. Exposure of cells expressingRUP25 to(5-hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanone isshown by Applicant to lower the level of intracellular cAMP.

Applicant has identified 1-Isopropyl-1H-benzotriazole-5-carboxylic acidto be an agonist for hRUP38 but not for RUP25. Exposure of cellsexpressing RUP38 to 1-Isopropyl-1H-benzotriazole-5-carboxylic acid isshown by Applicant to lower the level of intracellular cAMP. Exposure ofRUP38 within adipocyte primary cultures derived from human subcutaneousfat to 1-Isopropyl-1H-benzotriazole-5-carboxylic acid is shown byApplicant to inhibit lipolysis.

Applicant has identified 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid toalso be an agonist for RUP38 but not for either RUP25 or RUP19. Exposureof cells expressing RUP38 to 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid isshown by Applicant to lower the level of intracellular cAMP.

Applicant, supra, provides direct in vitro evidence for RUP25 and RUP38being antilipolytic and direct in vivo evidence in the rat for RUP25being antilipolytic. Applicant also notes illustrative clinical evidencethat nicotinic acid receptor is antilipolytic. Said evidence isconsistent with the disclosure by Applicant in the present applicationthat RUP25 is a nicotinic acid and an antilipolytic GPCR. Said evidenceis consistent with the disclosure by Applicant in the presentapplication that RUP38 and RUP19 are antilipolytic GPCRs. Said evidenceis consistent with the disclosure by Applicant in the presentapplication that RUP11 is an antilipolytic GPCR.

RUP38, RUP19 and RUP11 are further disclosed herein as beingantilipolytic GPCRs responsive to agonists other than nicotinic acid.The failure of nicotinic acid to serve as an agonist for RUP38, RUP19 orRUP11 indicates that RUP38, RUP19 and RUP11 represent novelantilipolytic pathways not engaged by conventional nicotinic acidtherapy.

See, Table M (Example 24) for a brief Summary and other additionalExamples, infra.

In a first aspect, the invention features a method of identifyingwhether a candidate compound is a modulator of a nicotinic acid GPCR,said receptor comprising an amino acid sequence selected from the groupconsisting of:

(a) SEQ. ID. NO.:36 (hRUP25);

(b) SEQ. ID. NO.:137 (mRUP25); and

(c) SEQ. ID. NO.:139 (rRUP25);

or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence;

comprising the steps of:

(a′) contacting the candidate compound with the receptor;

(b′) determining whether the receptor functionality is modulated;

wherein a change in receptor functionality is indicative of thecandidate compound being a modulator of a nicotinic acid GPCR.

In some embodiments, said nicotinic acid GPCR is endogenous.

In some preferred embodiments, said nicotinic acid GPCR is recombinant.

Preferred said identified modulator binds to said GPCR.

In some embodiments, said contacting is carried out in the presence of aknown ligand of the GPCR. In some embodiments, said known ligand is anagonist of the GPCR. In some embodiments, said agonist is nicotinic acidor an analog or derivative thereof. In some embodiments, said agonist is(−)-nicotine or an analog or derivative thereof.

The invention also relates to a method of identifying whether acandidate compound is a modulator of lipolysis, comprising the steps of:

-   -   (a) contacting the candidate compound with a GPCR comprising an        amino acid sequence selected from the group consisting of:        -   (i) SEQ. ID. NO.:36 (hRUP25);        -   (ii) SEQ. ID. NO.:137 (mRUP25); and        -   (iii) SEQ. ID. NO.:139 (rRUP25);        -   or an allelic variant, a biologically active mutant, or a            biologically active fragment of said amino acid sequence;            and    -   (b) determining whether the receptor functionality is modulated;        wherein a change in receptor functionality is indicative of the        candidate compound being a modulator of lipolysis.

In some embodiments, said GPCR is endogenous.

In some preferred embodiments, said GPCR is recombinant.

Preferred said identified modulator binds to said GPCR.

In some embodiments, said contacting is carried out in the presence of aknown ligand of the GPCR. In some embodiments, said known ligand is anagonist of the GPCR. In some embodiments, said agonist is nicotinic acidor an analog or derivative thereof. In some embodiments, said agonist is(−)-nicotine or an analog or derivative thereof.

The invention also relates to a method of determining whether acandidate compound is a modulator of a nicotinic acid GPCR, comprisingthe steps of:

-   -   (a) culturing nicotinic acid GPCR-expressing host cells under        conditions that would allow expression of a recombinant        nicotinic acid GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant nicotinic acid GPCR        comprising an amino acid sequence selected from the group        consisting of:        -   (i) SEQ. ID. NO.:36 (hRUP25);        -   (ii) SEQ. ID. NO.:137 (mRUP25); and        -   (iii) SEQ. ID. NO.:139 (rRUP25);        -   or an allelic variant, a biologically active mutant, or a            biologically active fragment of said amino acid sequence;    -   (b) contacting the nicotinic acid GPCR-expressing host cells of        step (a) with the candidate compound;    -   (c) contacting control host cells with the candidate compound of        step (b), wherein said control host cells do not express        recombinant nicotinic acid GPCR protein;    -   (d) measuring the modulating effect of the candidate compound        which interacts with the recombinant nicotinic acid GPCR from        the host cells of step (a) and control host cells of step (c);        and    -   (e) comparing the modulating effect of the test compound on the        host cells and control host cells.

The invention also relates to a method of determining whether acandidate compound is a modulator of a nicotinic acid GPCR, comprisingthe steps of:

-   -   (a) culturing nicotinic acid GPCR-expressing host cells under        conditions that would allow expression of a recombinant        nicotinic acid GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant nicotinic acid GPCR        comprising an amino acid sequence selected from the group        consisting of:        -   (i) SEQ. ID. NO.:36 (hRUP25);        -   (ii) SEQ. ID. NO.:137 (mRUP25); and        -   (iii) SEQ. ID. NO.:139 (rRUP25);        -   or an allelic variant, a biologically active mutant, or a            biologically active fragment of said amino acid sequence;    -   (b) contacting a first population of nicotinic acid        GPCR-expressing cells of step (a) with a known ligand of said        nicotinic receptor GPCR;    -   (c) contacting a second population of nicotinic acid        GPCR-expressing cells of step (a) with the candidate compound        and with the known nicotinic acid GPCR ligand;    -   (d) contacting control host cells with the candidate compound of        step (c), wherein said control host cells do not express        recombinant nicotinic acid GPCR protein;    -   (e) measuring the modulating effect of the candidate compound,        which interacts with recombinant nicotinic acid GPCR, in the        presence and absence of the known nicotinic acid GPCR ligand,        from the cells of step (b), step (c) and step (d); and    -   (f) comparing the modulating effect of the candidate compound as        determined from step (b), step (c) and step (d).

In some embodiments, said ligand is an agonist of said nicotinic acidGPCR. In a particular embodiment, said agonist is nicotinic acid or ananalog or derivative thereof. In other particular embodiment, saidagonist is (−)-nicotine or an analog or derivative thereof.

The invention also relates to a method of determining whether acandidate compound is a modulator of a nicotinic acid GPCR, comprisingthe steps of:

-   -   (a) culturing nicotinic acid GPCR-expressing host cells under        conditions that would allow expression of a recombinant        nicotinic acid GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant nicotinic acid GPCR        comprising an amino acid sequence selected from the group        consisting of:        -   (i) SEQ. ID. NO.:36 (hRUP25);        -   (ii) SEQ. ID. NO.:137 (mRUP25); and        -   (iii) SEQ. ID. NO.:139 (rRUP25);        -   or an allelic variant, a biologically active mutant, or a            biologically active fragment of said amino acid sequence;    -   (b) contacting a first population of the nicotinic acid        GPCR-expressing host cells of step (a) with the candidate        compound;    -   (c) not contacting a second population of the nicotinic acid        GPCR-expressing cells of step (a) with the candidate compound of        step (b);    -   (d) contacting control host cells to the candidate compound of        step (b), wherein said control host cells do not express        recombinant nicotinic acid GPCR protein;    -   (e) measuring the modulating effect of the candidate compound,        which interacts with recombinant nicotinic acid GPCR protein,        from the cells of step (b) and step (c) and from the cells of        step (d); and    -   (f) comparing the modulating effect of the candidate compound as        determined from step (b) and step (c) and from step (d).

In some embodiments, the nicotinic acid GPCR has an amino acid sequenceselected from the group consisting of:

SEQ. ID. NO.:36 (hRUP25);

-   -   (a) SEQ. ID. NO.:137 (mRUP25); and    -   (b) SEQ. ID. NO.:139 (rRUP25);    -   or an allelic variant, a biologically active mutant, or a        biologically active fragment of said amino acid sequence.

In some embodiments, the nicotinic acid GPCR comprises a biologicallyactive fragment of said amino acid sequence.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:36 further substituted at amino acid position 230 withlysine in place of isoleucine. In preferred embodiments, said EFA mutanthas the amino acid sequence of SEQ. ID. NO.:159.

In preferred embodiments, said G protein is Gi.

In other preferred embodiments, said determining is through the use of aMelanophore assay.

In other preferred embodiments, said determining is through themeasurement of the level of a second messenger selected from the groupconsisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositoltriphosphate (IP₃), diacylglycerol (DAG), and Ca²⁺. In further preferredembodiments, said second messenger is cAMP. In more preferredembodiments, the level of the cAMP is reduced. In some embodiments, saidmeasurement of cAMP is carried out with membrane comprising said GPCR.

In other preferred embodiments, said determining is through themeasurement of an activity up-regulated or down-regulated by a reductionin intracellular cAMP level. In further preferred embodiments, saiddown-regulated activity is intracellular lipolysis. In other furtherpreferred embodiments, said down-regulated activity is hormone sensitivelipase activity. In other further preferred embodiments, saidup-regulated activity is adiponectin secretion.

In other preferred embodiments, said determining is through CRE-reporterassay. In preferred embodiments, said reporter is luciferase. In someembodiments, said reporter is β-galactosidase.

In other embodiments, said recombinant host cell further comprisespromiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit and saiddetermining is through measurement of intracellular Ca²⁺. In preferredembodiments, said Ca²⁺ measurement is carried out by FLIPR.

In other embodiments, said recombinant host cell further comprisespromiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit and saiddetermining is through measurement of intracellular IP₃.

In other preferred embodiments, said determining is through themeasurement of GTPγS binding to membrane comprising said GPCR. Infurther preferred embodiments, said GTPγS is labeled with [³⁵S].

In other preferred embodiments, said method further comprises the stepof comparing the modulation of the receptor caused by the candidatecompound to a second modulation of the receptor caused by contacting thereceptor with a known modulator of the receptor. In some preferredembodiments, said known modulator is an agonist. In some preferredembodiments, said agonist is nicotinic acid or an analog or derivativethereof. In some preferred embodiments, said agonist is (−)-nicotine oran analog or derivative thereof.

In a second aspect, the invention features a modulator of a nicotinicacid GPCR identified according to the method of the first aspect,provided that the modulator is not identical to a compound having aformula selected from the group consisting of:

wherein:

R₁ is selected from the group consisting of halogen, hydroxyl,acetylamino, ammo, alkoxy, carboalkoxy, alkylthio, monoalkylamino,dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfonyl, saidalkyl groups containing from 1 to 4 carbons, trifluoromethyl,trifluoromethoxy, trifluoromethylthio, methoxymethyl, carboxy, carbamyl,alkanoyloxy containing up to 4 carbon atoms, phenyl, p-chlorophenyl,p-methylphenyl and p-aminophenyl;

R₂ is selected from the group consisting of halogen, alkannoyloxycontaining from 1-4 carbon atoms, carboalkoxy containing from 2 to 5carbon atoms, carbamyl, N-alkyl carbamyl and N,N-dialkylcarbamyl whereinsaid alkyl groups contain from 1-4 carbon atoms and trifluoromethyl;

n is a whole number from 0 to 4; and

N-oxides thereof;

R₃ and R₄ are hydrogen, alkyl containing from 1 to 4 carbon atoms orcycloalkyl containing from 3 to 7 carbon atoms;

n is a whole number from 0 to 4; and

N-oxides thereof;

wherein:

R₅ and R₆ are each selected from the group consisting of H, halogen,hydroxyl, amino, alkyloxy, alkylthio, monoalkylamino, dialkylamino,N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfoxy, alkylsulfony, saidalkyl groups containing from 1 to 4 carbons, trifluoromethyl,trifluoromethoxy, trifluoromethylthio, carboxy, carbamyl, alkanoyloxycontaining up to 4 carbon atoms, phenyl, p-chlorophenyl, p-methylphenyland p-aminophenyl;

n is a whole number from 0 to 4; and

N-oxides thereof;

wherein:

at least one of R₇, R₈ and R₉ is C₁₋₆ alkyl and the others are hydrogenatoms; R₁₀ is hydroxy or C₁₋₆ alkoxy, or a salt of the compounds whereinR₄ is hydoxy with a pharmaceutically acceptable base; and a 4-N-oxidethereof. The position of the N-oxide is designated by the followingnumbering and a structure for a 4-N-oxide has the following structure:

One particular 4-N-oxide is 5-Methylpyrazine-2-carboxylic acid-4-oxide(Acipimox™) and has the structure:

wherein:

at least one of R₇, R₈ and R₉ is C₁₋₆ alkyl and the others are hydrogenatoms; each of R₁₁ and R₁₂, which may be the same or different, ishydrogen orC₁₋₆ alkyl; and a 4-N-oxide thereof; the position of the N-oxide is thesame as described above herein;

wherein:

at least one of R₁₃ represents an alkyl group of 7-11 carbon atoms andR₁₄ represents H or a lower alkyl group of up to two carbon atoms, and apharmaceutically acceptable carrier;(g) Pyrazine-2-carboxylic acid amide and has the structure:

5-chloro-pyrazine-2-carboxylic acid amide and has the structure:

5-amino-pyrazine-2-carboxylic acid amide and has the structure:

5-benzyl-pyrazine-2-carboxylic acid amide and has the structure:

6-chloro-pyrazine-2-carboxylic acid amide and has the structure:

6-methoxy-pyrazine-2-carboxylic acid amide and has the structure:

3-chloro-pyrazine-2-carboxylic acid amide and has the structure:

3-methoxy-pyrazine-2-carboxylic acid amide and has the structure:

pyrazine-2-carboxylic acid ethylamide and has the structure:

morpholin-4-yl-pyrazine-2-ylmethanone and has the structure:

5-methyl-pyrazine-2-carboxylic acid (6-methyl-pyrazin-2-yl)-amide andhas the structure:

5-methyl-pyrazine-2-carboxylic acid (5-methyl-pyrazin-2-yl)-amide andhas the structure:

5-methyl-pyrazine-2-carboxylic acid (3-methyl-pyrazin-2-yl)-amide andhas the structure:

(5-methyl-pyrazin-2-yl)-morpholin-4-yl-methanone and has the structure:

5-methyl-pyrazine-2-carboxylic acid hydroxyamide and has the structure:

pyrazine-2-carboxylic acid and has the structure:

5-amino-pyrazine-2-carboxylic acid and has the structure:

5-benzyl-pyrazine-2-carboxylic acid and has the structure:

6-chloro-pyrazine-2-carboxylic acid and has the structure:

6-methoxy-pyrazine-2-carboxylic acid and has the structure:

3-hydroxy-pyrazine-2-carboxylic acid and has the structure:

5-methyl-pyrazine-2-carboxylic acid 2-hydroxy-ethyl ester and has thestructure:

5-methyl-pyrazine-2-carboxylic acid allyl ester and has the structure:

5-methyl-pyrazine-2-carboxylic acid phenyl ester and has the structure:

5-methyl-pyrazine-2-carboxylic acid ethoxycarbonylmethyl ester and hasthe structure:

pyrazine-2-carboxylic acid methyl ester and has the structure:

2-methyl-5-(1H-tetrazol-5-yl)-pyrazine and has the structure:

and 4-N-oxides thereof as described above herein;(h) 5-(3-(5-Methyl)isoxazolyl)tetrazole and has the structure:

(i) 5-(5-(3-Methyl)isoxazolyl)tetrazole and has the structure:

(j) 5-(3-Quinolyl)tetrazole and has the structure:

(k) Nicotinic acid and has the structure:

(l) Pyridazine-4-carboxylic acid and has the structure:

(m) 3-Pyridine acetic acid and has the structure:

(n) 5-Methylnicotinic acid and has the structure:

(o) 6-Methylnicotinic acid and has the structure:

(p) Nicotinic acid-1-oxide and has the structure:

(q) 2-Hydroxynicotinic acid and has the structure:

(r) Furane-3-carboxylic acid and has the structure:

(s) 5-Methylpyrazole-3-carboxylic acid and has the structure:

t) 3-Methylisoxazole-5-carboxylic acid and has the structure:

In preferred embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.More preferably, said modulator is an agonist. In some embodiments, saidmodulator is a partial agonist.

In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 1000 μM in GTPγS binding assay carried out with membrane fromstably transfected CHO cells expressing recombinant hRUP25 polypeptidehaving the amino acid sequence of SEQ. ID. NO.:36. In some embodiments,said modulator is an agonist with an EC₅₀ of less than 900 μM in saidassay. In some embodiments, said modulator is an agonist with an EC₅₀ ofless than 800 μM in said assay. In some embodiments, said modulator isan agonist with an EC₅₀ of less than 700 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 600μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 550 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 500 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 450 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 400 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 350μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 300 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 250 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 200 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 150 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 100μM in said assay. In some preferred embodiments, said modulator is anagonist with an EC₅₀ in said assay of less than a value selected fromthe interval of 600 μM to 1000 μM.

In some embodiments, said modulator is selective for the GPCR.

In some embodiments, said modulator is antilipolytic.

In some embodiments, said modulator is orally bioavailable. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45 % relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In highly less preferred embodiments, said modulator is an antibody orderivative thereof.

In a third aspect, the invention features the method of the firstaspect, wherein said candidate compound is an agonist of hRUP38 GPCRcomprising the amino acid sequence of SEQ. ID. NO.:135 and wherein saidmethod further comprises the step of comparing the modulation of hRUP25GPCR comprising the amino acid sequence of SEQ. ID. NO.:36 caused bysaid agonist to a second modulation of hRUP25 GPCR comprising a variantof said amino acid sequence caused by contacting the variant hRUP25 GPCRwith said agonist.

In preferred embodiments, said variant amino acid sequence is identicalto the amino acid sequence of SEQ. ID. NO.:36, further comprising asingle amino acid substitution selected from the group consisting of:

(a) A for V at amino acid position 27 of SEQ. ID. NO.:36;

(b) V for L at amino acid position 83 of SEQ. ID. NO.:36;

(c) Y for N at amino acid position 86 of SEQ. ID. NO.:36;

(d) S for W at amino acid position 91 of SEQ. ID. NO.:36;

(e) N for K at amino acid position 94 of SEQ. ID. NO.:36;

(f) V for M at amino acid position 103 of SEQ. ID. NO.:36;

(g) F for L at amino acid position 107 of SEQ. ID. NO.:36;

(h) W for R at amino acid position 142 of SEQ. ID. NO.:36;

(i) V for 1 at amino acid position 156 of SEQ. ID. NO.:36;

(j) L for M at amino acid position 167 of SEQ. ID. NO.:36;

(k) L for P at amino acid position 168 of SEQ. ID. NO.:36;

(l) P for G at amino acid position 173 of SEQ. ID. NO.:36;

(m) V for L at amino acid position 176 of SEQ. ID. NO.:36;

(n) I for S at amino acid position 178 of SEQ. ID. NO.:36;

(O)R for Q at amino acid position 187 of SEQ. ID. NO.:36;

(p) L for F at amino acid position 198 of SEQ. ID. NO.:36; and

(q) N for P at amino acid position 363 of SEQ. ID. NO.:36.

In particularly preferred embodiments, said method is used to identifywhether said substituted amino acid additionally found at the identicalposition within SEQ. ID. NO.:135 is necessary for modulation of saidhRUP38 GPCR by said agonist, comprising the steps of:

(a) determining the level of modulation of said hRUP25 GPCR by saidagonist; and

(b) determining the level of modulation of said variant hRUP25 GPCR bysaid agonist; wherein if said level of modulation for (b) is greaterthan said level of modulation for (a), then said substituted amino acidis necessary for modulation of said hRUP38 GPCR by said agonist.

In a fourth aspect, the invention features a method of modulating theactivity of a nicotinic acid GPCR, said receptor comprising an aminoacid sequence selected from the group consisting of:

(a) SEQ. ID. NO.:36 (hRUP25);

(b) SEQ. ID. NO.:137 (mRUP25); and

(c) SEQ. ID. NO.:139 (rRUP25);

or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence;

comprising the step of contacting the receptor with the modulator of thesecond aspect.

In some embodiments, the nicotinic acid GPCR has an amino acid sequenceselected from the group consisting of:

(a) SEQ. ID. NO.:36 (hRUP25);

(b) SEQ. ID. NO.:137 (mRUP25); and

(c) SEQ. ID. NO.:139 (rRUP25);

or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence.

In some embodiments, the nicotinic acid GPCR comprises an activefragment of said amino acid sequence.

In some embodiments, the nicotinic acid GPCR is endogenous.

In some embodiments, the nicotinic acid GPCR is recombinant.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:36 further substituted at amino acid position 230 withlysine in place of isoleucine. In preferred embodiments, said EFA mutanthas the amino acid sequence of SEQ. ID. NO.:159.

In preferred embodiments, said G protein is Gi.

In some preferred embodiments, said modulator is an agonist.

In preferred embodiments, said modulator is selective for the GPCR.

In other preferred embodiments, said contacting comprises administrationof the modulator to a membrane comprising the receptor.

In other preferred embodiments, said contacting comprises administrationof the modulator to a cell or tissue comprising the receptor.

In other preferred embodiments, said contacting comprises administrationof the modulator to an individual comprising the receptor. In morepreferred embodiments, said individual is a mammal. In other morepreferred embodiments, said mammal is a horse, cow, sheep, pig, cat,dog, rabbit, mouse, rat, non-human primate or human. Yet more preferredis mouse, rat or human. Most preferred is human.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP25polypeptide having the amino acid sequence of SEQ. ID. NO.:36. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said administration is oral.

In preferred embodiments, said modulator is an agonist and saidindividual is in need of prevention of or treatment for ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said modulator is an inverse agonist and saidmetabolic-related disorder relates to a low level of plasma free fattyacids.

In other preferred embodiments, said modulator is an agonist and saidindividual is in need of a change in lipid metabolism selected from thegroup consisting of:

(a) a decrease in the level of plasma triglycerides;

(b) a decrease in the level of plasma free fatty acids;

(c) a decrease in the level of plasma cholesterol;

(d) a decrease in the level of LDL-cholesterol;

(e) an increase in the level of HDL-cholesterol;

(f) a decrease in the total cholesterol/HDL-cholesterol ratio; and

(g) an increase in the level of plasma adiponectin.

In other preferred embodiments, said needed change in lipid metabolismis a decrease in the postprandial increase in plasma free fatty acidsdue to a high fat meal or an inhibition of the progression from impairedglucose tolerance to insulin resistance.

In some embodiments, the modulator is an inverse agonist and the neededchange in lipid metabolism is an increase in the level of plasma freefatty acids.

In other preferred embodiments, said modulator is an agonist and saidindividual is a mouse genetically predisposed to a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure.

In further preferred embodiments, said metabolic-related disorder ishyperlipidemia.

In further preferred embodiments, said method is used to identifywhether said agonist has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering said agonist to the mouse; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering said agonist compared to not administeringsaid agonist; wherein said determination is indicative of said agonisthaving therapeutic efficacy.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other preferred embodiments, said modulator is an agonist and saidindividual is a rat genetically predisposed to a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Infurther preferred embodiments, said metabolic-related disorder ishyperlipidemia.

In further preferred embodiments, said method is used to identifywhether said agonist has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes; comprising the steps of:

(a′) administering or not administering said agonist to the rat; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering said agonist compared to not administeringsaid agonist; wherein said determination is indicative of said agonisthaving therapeutic efficacy.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In a fifth aspect, the invention features a method of preventing ortreating a disorder of lipid metabolism in an individual comprisingcontacting a therapeutically effective amount of the modulator of thesecond aspect with a nicotinic acid GPCR, said receptor comprising anamino acid sequence selected from the group consisting of:

(a) SEQ. ID. NO.:36 (hRUP25);

(b) SEQ. ID. NO.:137 (mRUP25); and

(c) SEQ. ID. NO.:139 (rRUP25);

or an allelic variant or biologically active fragment of said amino acidsequence.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP25polypeptide having the amino acid sequence of SEQ. ID. NO.:36. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said contacting comprises oraladministration of said modulator to said individual.

In preferred embodiment, said modulator is an agonist and said disorderof lipid metabolism is selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In a sixth aspect, the invention features a method of preventing ortreating a metabolic-related disorder in an individual comprisingcontacting a therapeutically effective amount of the modulator of thesecond aspect with a nicotinic acid GPCR, said receptor comprising anamino acid sequence selected from the group consisting of:

(a) SEQ. ID. NO.:36 (hRUP25);

(b) SEQ. ID. NO.:137 (mRUP25); and

(c) SEQ. ID. NO.:139 (rRUP25);

or an allelic variant or biologically active fragment of said amino acidsequence.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP25polypeptide having the amino acid sequence of SEQ. ID. NO.:36. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said contacting comprises oraladministration of said modulator to said individual.

In preferred embodiment, said modulator is an agonist and saidmetabolic-related disorder is selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In a seventh aspect, the invention features a method of preparing acomposition which comprises identifying a modulator of a nicotinic acidGPCR and then admixing a carrier and the modulator, wherein themodulator is identifiable by the method of the first aspect and providedthat the modulator is not identical to a compound having a formulaselected from the group consisting of:

wherein:

R₁ is selected from the group consisting of halogen, hydroxyl,acetylamino, amino, alkoxy, carboalkoxy, alkylthio, monoalkylamino,dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfonyl, saidalkyl groups containing from 1 to 4 carbons, trifluoromethyl,trifluoromethoxy, trifluoromethylthio, methoxymethyl, carboxy, carbamyl,alkanoyloxy containing up to 4 carbon atoms, phenyl, p-chlorophenyl,p-methylphenyl and p-aminophenyl;

R₂ is selected from the group consisting of halogen, alkannoyloxycontaining from 1-4 carbon atoms, carboalkoxy containing from 2 to 5carbon atoms, carbamyl, N-alkyl carbamyl and N,N-dialkylcarbamyl whereinsaid alkyl groups contain from 1-4 carbon atoms and trifluoromethyl;

n is a whole number from 0 to 4; and

N-oxides thereof;

R₃ and R₄ are hydrogen, alkyl containing from 1 to 4 carbon atoms orcycloalkyl containing from 3 to 7 carbon atoms;

n is a whole number from 0 to 4; and

N-oxides thereof;

wherein:

R₅ and R₆ are each selected from the group consisting of H, halogen,hydroxyl, amino, alkyloxy, alkylthio, monoalkylamino, dialkylamino,N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfoxy, alkylsulfony, saidalkyl groups containing from 1 to 4 carbons, trifluoromethyl,trifluoromethoxy, trifluoromethylthio, carboxy, carbamyl, alkanoyloxycontaining up to 4 carbon atoms, phenyl, p-chlorophenyl, p-methylphenyland p-aminophenyl;

n is a whole number from 0 to 4; and

N-oxides thereof;

wherein:

at least one of R₇, R₈ and R₉ is C₁₋₆ alkyl and the others are hydrogenatoms; R₁₀ is hydroxy or C₁₋₆ alkoxy, or a salt of the compounds whereinR₄ is hydoxy with a pharmaceutically acceptable base; and a 4-N-oxidethereof. The position of the N-oxide is designated by the followingnumbering and a structure for a 4-N-oxide has the following structure:

One particular 4-N-oxide is 5-Methylpyrazine-2-carboxylic acid-4-oxide(Acipimox™) and has the structure:

wherein:

at least one of R₇, R₈ and R₉ is C₁₋₆ alkyl and the others are hydrogenatoms; each of R₁₁ and R₁₂, which may be the same or different, ishydrogen orC₁₋₆ alkyl; and a 4-N-oxide thereof; the position of the N-oxide is thesame as described above herein;

wherein:

at least one of R₁₃ represents an alkyl group of 7-11 carbon atoms andR₁₄ represents H or a lower alkyl group of up to two carbon atoms, and apharmaceutically acceptable carrier;(g) Pyrazine-2-carboxylic acid amide and has the structure:

5-chloro-pyrazine-2-carboxylic acid amide and has the structure:

5-amino-pyrazine-2-carboxylic acid amide and has the structure:

5-benzyl-pyrazine-2-carboxylic acid amide and has the structure:

6-chloro-pyrazine-2-carboxylic acid amide and has the structure:

6-methoxy-pyrazine-2-carboxylic acid amide and has the structure:

3-chloro-pyrazine-2-carboxylic acid amide and has the structure:

3-methoxy-pyrazine-2-carboxylic acid amide and has the structure:

pyrazine-2-carboxylic acid ethylamide and has the structure:

morpholin-4-yl-pyrazine-2-ylmethanone and has the structure:

5-methyl-pyrazine-2-carboxylic acid (6-methyl-pyrazin-2-yl)-amide andhas the structure:

5-methyl-pyrazine-2-carboxylic acid (5-methyl-pyrazin-2-yl)-amide andhas the structure:

5-methyl-pyrazine-2-carboxylic acid (3-methyl-pyrazin-2-yl)-amide andhas the structure:

(5-methyl-pyrazin-2-yl)-morpholin-4-yl-methanone and has the structure:

5-methyl-pyrazine-2-carboxylic acid hydroxyamide and has the structure:

pyrazine-2-carboxylic acid and has the structure:

5-amino-pyrazine-2-carboxylic acid and has the structure:

5-benzyl-pyrazine-2-carboxylic acid and has the structure:

6-chloro-pyrazine-2-carboxylic acid and has the structure:

6-methoxy-pyrazine-2-carboxylic acid and has the structure:

3-hydroxy-pyrazine-2-carboxylic acid and has the structure:

5-methyl-pyrazine-2-carboxylic acid 2-hydroxy-ethyl ester and has thestructure:

5-methyl-pyrazine-2-carboxylic acid allyl ester and has the structure:

5-methyl-pyrazine-2-carboxylic acid phenyl ester and has the structure:

5-methyl-pyrazine-2-carboxylic acid ethoxycarbonylmethyl ester and hasthe structure:

pyrazine-2-carboxylic acid methyl ester and has the structure:

2-methyl-5-(1H-tetrazol-5-yl)-pyrazine and has the structure:

and 4-N-oxides thereof as described above herein;(h) 5-(3-(5-Methyl)isoxazolyl)tetrazole and has the structure:

(i) 5-(5-(3-Methyl)isoxazolyl)tetrazole and has the structure:

(j) 5-(3-Quinolyl)tetrazole and has the structure:

(k) Nicotinic acid and has the structure:

(l) Pyridazine-4-carboxylic acid and has the structure:

(m) 3-Pyridine acetic acid and has the structure:

(n) 5-Methylnicotinic acid and has the structure:

(O) 6-Methylnicotinic acid and has the structure:

(p) Nicotinic acid-1-oxide and has the structure:

(q) 2-Hydroxynicotinic acid and has the structure:

(r) Furane-3-carboxylic acid and has the structure:

(s) 5-Methylpyrazole-3-carboxylic acid and has the structure:

t) 3-Methylisoxazole-5-carboxylic acid and has the structure:

In preferred embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.More preferably, said modulator is an agonist. In some embodiments, saidmodulator is a partial agonist.

In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 1000 μM in GTPγS binding assay carried out with membrane fromstably transfected CHO cells expressing recombinant hRUP25 polypeptidehaving the amino acid sequence of SEQ. ID. NO.:36. In some embodiments,said modulator is an agonist with an EC₅₀ of less than 900 μM in saidassay. In some embodiments, said modulator is an agonist with an EC₅₀ ofless than 800 μM in said assay. In some embodiments, said modulator isan agonist with an EC₅₀ of less than 700 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 600μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 550 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 500 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 450 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 400 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 350μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 300 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 250 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 200 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 150 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 100μM in said assay. In some preferred embodiments, said modulator is anagonist with an EC₅₀ in said assay of less than a value selected fromthe interval of 600 μM to 1000 μM.

In some embodiments, said modulator is selective for the GPCR.

In some embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to eitherintraperitoneal or intravenous administration. In some preferredembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to either intraperitoneal orintravenous administration.

In an eighth aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of the modulator of the second aspect. Inpreferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP25polypeptide having the amino acid sequence of SEQ. ID. NO.:36. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In a ninth aspect, the invention features a method of changing lipidmetabolism comprising providing or administering to an individual inneed of said change said pharmaceutical or physiologically acceptablecomposition of the eighth aspect, said needed change in lipid metabolismselected from the group consisting of:

(a) a decrease in the level of plasma triglycerides;

(b) a decrease in the level of plasma free fatty acids;

(c) a decrease in the level of plasma cholesterol;

(d) a decrease in the level of LDL-cholesterol;

(e) an increase in the level of HDL-cholesterol;

(f) a decrease in the total cholesterol/HDL-cholesterol ratio; and

(g) an increase in the level of plasma adiponectin.

In preferred embodiments, a therapeutically effective amount of saidpharmaceutical or physiologically acceptable composition is provided oradministered to said individual.

In some preferred embodiments, said providing or administering of saidpharmaceutical or physiologically acceptable composition is oral.

In other preferred embodiments, said needed change in lipid metabolismis a decrease in the postprandial increase in plasma free fatty acidsdue to a high fat meal or an inhibition of the progression from impairedglucose tolerance to insulin resistance.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In a tenth aspect, the invention features a method of preventing ortreating a metabolic-related disorder comprising providing oradministering to an individual in need of said treatment saidpharmaceutical or physiologically acceptable composition of the eighthaspect, said metabolic-related disorder selected from the groupconsisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In preferred embodiments, a therapeutically effective amount of saidpharmaceutical or physiologically acceptable composition is provided oradministered to said individual.

In some preferred embodiments, said providing or administering of saidpharmaceutical or physiologically acceptable composition is oral.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, stroke, Syndrome X, andheart disease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In an eleventh aspect, the invention features a method of using themodulator of the second aspect for the preparation of a medicament forthe treatment of a disorder in lipid metabolism in an individual.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP25polypeptide having the amino acid sequence of SEQ. ID. NO.:36. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said treatment comprises oraladministration of said medicament to said individual.

In preferred embodiments, said modulator is an agonist and said disorderin lipid metabolism is selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In a twelfth aspect, the invention features a method of using themodulator of the second aspect for the preparation of a medicament forthe treatment of a metabolic-related disorder in an individual.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP25polypeptide having the amino acid sequence of SEQ. ID. NO.:36. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said treatment comprises oraladministration of said medicament to said individual.

In preferred embodiments, said modulator is an agonist and saidmetabolic-related disorder is selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In a thirteenth aspect, the invention features a method of identifyingwhether a candidate compound binds to a nicotinic acid GPCR, saidreceptor comprising an amino acid sequence selected from the groupconsisting of:

(a) SEQ. ID. NO.:36 (hRUP25);

(b) SEQ. ID. NO.:137 (mRUP25); and

(c) SEQ. ID. NO.:139 (rRUP25);

or an allelic variant or a biologically active fragment of said aminoacid sequence; comprising the steps of:

(a′) contacting the receptor with a labeled reference compound known tobind to the GPCR in the presence or absence of the candidate compound;and

(b′) determining whether the binding of said labeled reference compoundto the receptor is inhibited in the presence of the candidate compound;

wherein said inhibition is indicative of the candidate compound bindingto a nicotinic acid GPCR.

In some embodiments, the nicotinic acid GPCR comprises a biologicallyactive fragment of said amino acid sequence.

In some embodiments, the nicotinic acid GPCR is endogenous.

In preferred embodiments, the nicotinic acid GPCR is recombinant.

In preferred embodiments, said G protein is Gi.

In some preferred embodiments, said reference compound is nicotinicacid.

In some preferred embodiments, said reference compound is (−)-nicotine.

In some preferred embodiments, said reference compound is the modulatorof the second aspect.

In other embodiments, said reference compound is an antibody specificfor the GPCR, or a derivative thereof.

In preferred embodiments, said reference compound comprises a labelselected from the group consisting of:

(a) radioisotope;

(b) enzyme; and

(c) fluorophore.

In some preferred embodiments, said label is ³H.

In other embodiments, said method further comprises the step ofcomparing the level of inhibition of binding of a labeled firstreference compound by the candidate compound to a second level ofinhibition of binding of said labeled first reference compound by asecond reference compound known to bind to the GPCR.

In a fourteenth aspect, the invention features a method of making amouse genetically predisposed to a metabolic-related disorder selectedfrom the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the step of knocking out the gene encoding the nicotinic acidmRUP25 GPCR polypeptide of SEQ. ID. NO.:137.

In some preferred embodiments, said knocking out the gene encoding thenicotinic acid mRUP25 GPCR polypeptide pf SEQ. ID. NO.:137 isessentially restricted to adipocytes.

In a fifteenth aspect, the invention features the knockout mouseaccording to the method of the fourteenth aspect.

In a sixteenth aspect, the invention features a method of using theknockout mouse of the fifteenth aspect to identify whether a candidatecompound has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering the compound to the mouse; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering the compound compared to not administeringthe compound; wherein said determination is indicative of the compoundhaving therapeutic efficacy.

In a seventeenth aspect, the invention features a method of making a ratgenetically predisposed to a metabolic-related disorder selected fromthe group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the step of knocking out the gene encoding the nicotinic acidrRUP25 GPCR polypeptide of SEQ. ID. NO.:139.

In some preferred embodiments, said knocking out the gene encoding thenicotinic acid rRUP25 GPCR polypeptide pf SEQ. ID. NO.:139 isessentially restricted to adipocytes.

In an eighteenth aspect, the invention features the knockout rataccording to the method of the seventeenth aspect.

In a nineteenth aspect, the invention features a method of using theknockout rat of the eighteenth aspect to identify whether a candidatecompound has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering the compound to the rat; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering the compound compared to not administeringthe compound; wherein said determination is indicative of the compoundhaving therapeutic efficacy.

In a twentieth aspect, the invention features an isolated, purified orrecombinant RUP25 polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising a contiguous span of at least 75nucleotides of SEQ.ID. NO.:35, SEQ. ID. NO.:136 or SEQ. ID. NO.:138, oran allelic variant of said polynucleotide;

(b) a polynucleotide comprising a contiguous span of at least 150nucleotides of SEQ. ID. NO.:35, SEQ. ID. NO.:136 or SEQ. ID. NO.:138, oran allelic variant of said polynucleotide;

(c) a polynucleotide comprising a contiguous span of at least 250nucleotides of SEQ. ID. NO.:35, SEQ. ID. NO.:136 or SEQ. ID. NO.:138, oran allelic variant of said polynucleotide;

(d) a polynucleotide comprising a contiguous span of at least 350nucleotides of SEQ. ID. NO.:35, SEQ. ID. NO.:136 or SEQ. ID. NO.:138, oran allelic variant of said polynucleotide;

(e) a polynucleotide comprising a contiguous span of at least 500nucleotides of SEQ. ID. NO.:35, SEQ. ID. NO.:136 or SEQ. ID. NO.:138, oran allelic variant of said polynucleotide;

(f) a polynucleotide comprising a contiguous span of at least 750nucleotides of SEQ. ID. NOs.:35, SEQ. ID. NO.:136 or SEQ. ID. NO.:138,or an allelic variant of said polynucleotide;

(g) a polynucleotide comprising a contiguous span of at least 1000nucleotides of SEQ. ID. NO.:35, SEQ. ID. NO.:136 or SEQ. ID. NO.:138, oran allelic variant of said polynucleotide;

(h) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 20 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO: 137 or SEQ.ID. NO.:139 or an allelic variant of said polypeptide;

(i) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 30 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ.ID. NO.:139 or an allelic variant of said polypeptide;

(j) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 40 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ.ID. NO.:139 or an allelic variant of said polypeptide;

(k) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 50 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ.ID. NO.:139 or an allelic variant of said polypeptide;

(l) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 75 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ.ID. NO.:139 or an allelic variant of said polypeptide;

(m) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 100 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ.ID. NO.:139 or an allelic variant of said polypeptide;

(n) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 150 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ.ID. NO.:139 or an allelic variant of said polypeptide;

(O) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 200 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ.ID. NO.:139 or an allelic variant of said polypeptide;

(p) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 250 amino acids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ.ID. NO.:139 or an allelic variant of said polypeptide; and

(q) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 300 amino acids of SEQ.ID. NO.:36, SEQ. ID. NO.:137 or SEQ.ID. NO.:139 or an allelic variant of said polypeptide.

The invention also relates to an isolated, purified or recombinant RUP25polynucleotide wherein said polynucleotide is selected from the groupconsisting of:

(a) a polynucleotide comprising a nucleotide sequence selected from thegroup consisting of SEQ. ID. NO.:35, SEQ. ID. NO.:136 and SEQ. ID.NO.:138 or an allelic variant of said polynucleotide;

(b) a polynucleotide selected from the group consisting of thepolynucleotide of SEQ. ID. NO.:35, the polynucleotide of SEQ. ID.NO.:136 and the polynucleotide of SEQ. ID. NO.:138, or an allelicvariant of said polynucleotide;

(c) a polynucleotide comprising a nucleotide sequence encoding apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ. ID. NO.:36, SEQ. ID. NO.:137 and SEQ. ID. NO.:139 oran allelic variant of said polypeptide; and

(d) a polynucleotide encoding a polypeptide having an amino acidsequence selected from the group consisting of SEQ. ID. NO.:36, SEQ. ID.NO.:137 and SEQ. ID. NO.:139 or an allelic variant of said polypeptide.

In preferred embodiments, said isolated, purified or recombinantpolynucleotide comprises at least 8 contiguous nucleotides of apolynucleotide of the present invention. In other preferred embodiments,said isolated, purified or recombinant polynucleotide comprises at least10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,500, 600, 700, 800, 900 or 1000 contiguous nucleotides of apolynucleotide of the present invention. Preferably said polynucleotideencodes full-length RUP25 polypeptide or a biologically active fragmentthereof.

The polynucleotides of the present invention include genomicpolynucleotides comprising RUP25 polynucleotides of the invention.

The present invention also relates to a polynucleotide encoding a fusionprotein, wherein said fusion protein comprises an RUP25 polypeptide ofthe invention fused to a heterologous polypeptide. In a preferredembodiment, said polypeptide of the invention is constitutively activeand said heterologous polypeptide is a G protein. In other embodiments,said heterologous polypeptide provides an antigenic epitope. In apreferred embodiment, said heterologous polypeptide provides ahemaglutinin (HA) antigenic epitope. Methods relating to apolynucleotide encoding a fusion protein are well known to those ofordinary skill in the art.

The polynucleotides of the present invention also include variantpolynucleotides at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%identical to an RUP25 polynucleotide of the invention. In a particularlypreferred embodiments, polynucleotide sequence homologies are evaluatedusing the Basic Local Alignment Search Tool (“BLAST”), which is wellknown in the art [See, e.g., Karlin and Altschul, Proc Natl Acad Sci USA(1990) 87:2264-8; Altschul et al., J Mol Biol (1990) 215:403-410;Altschul et al., Nature Genetics (1993) 3:266-72; and Altschul et al.,Nucleic Acids Res (1997) 25:3389-3402; the disclosures of which areincorporated by reference in their entirety].

In further preferred embodiments, the invention features the complementof said polynucleotide.

In a twenty-first aspect, the invention features an isolated, purifiedor recombinant RUP25 polypeptide selected from the group consisting of:

(a) a polypeptide comprising a contiguous span of at least 20 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(b) a polypeptide comprising a contiguous span of at least 30 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(c) a polypeptide comprising a contiguous span of at least 40 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(d) a polypeptide comprising a contiguous span of at least 50 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(e) a polypeptide comprising a contiguous span of at least 75 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(f) a polypeptide comprising a contiguous span of at least 100 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(g) a polypeptide comprising a contiguous span of at least 150 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(h) a polypeptide comprising a contiguous span of at least 200 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(i) a polypeptide comprising a contiguous span of at least 250 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139; and

(j) a polypeptide comprising a contiguous span of at least 300 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139; or anallelic variant of said polypeptide.

The invention also relates to an isolated, purified or recombinant RUP25polypeptide wherein said polypeptide is selected from the groupconsisting of:

(a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ. ID. NO.:36, SEQ. ID. NO.:137 and SEQ. ID.NO.:139; and

(b) a polypeptide selected from the group consisting of the polypeptideof SEQ. ID. NO.:36, the polypeptide of SEQ. ID. NO: 137 and thepolypeptide of SEQ. ID. NO.:139; or an allelic variant, a biologicallyactive mutant, or a biologically active fragment of said polypeptide.

In preferred embodiments, said isolated, purified or recombinantpolypeptide comprises at least 6 contiguous amino acids of an RUP25polypeptide of the invention. In further embodiments, said isolated,purified or recombinant polypeptide comprises at least 10, 12, 15, 20,25, 30, 35, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275 or300 contiguous amino acids of a polypeptide of the present invention.Preferably, said polypeptide is full-length RUP25 polypeptide or anactive fragment thereof.

The present invention also relates to a fusion protein, wherein saidfusion protein comprises an RUP25 polypeptide of the invention fused toa heterologous polypeptide. In a preferred embodiment, said polypeptideof the invention is constitutively active and said heterologouspolypeptide is a G protein. In other preferred embodiments, saidheterologous polypeptide provides an antigenic epitope. In particularlypreferred embodiment, said heterologous polypeptide provides ahemaglutinin (HA) antigenic epitope. Methods relating to a fusionprotein are well known to those of ordinary skill in the art.

The polypeptides of the present invention also include variantpolypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to an RUP25 polypeptide of the invention. In aparticularly preferred embodiments, polypeptide sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”), whichis well known in the art [See, e.g., Karlin and Altschul, Proc Natl AcadSci USA (1990) 87:2264-8; Altschul et al., J Mol Biol (1990) 215:403410;Altschul et al., Nature Genetics (1993) 3:266-72; and Altschul et al.,Nucleic Acids Res (1997) 25:3389-3402; the disclosures of which areincorporated by reference in their entirety].

In an twenty-second aspect, the invention features a compositioncomprising, consisting essentially of, or consisting of the RUP25polypeptide of the twenty-first aspect.

In a twenty-third aspect, the invention features a recombinant vector,said vector comprising, consisting essentially of, or consisting of thepolynucleotide of the twentieth aspect. In some preferred embodiments,said vector is a targeting vector used in a method of inactivating agene encoding a nicotinic acid GPCR of the invention. In other preferredembodiments, said vector is used in a method of transient or stabletransfection.

In particularly preferred embodiment, said vector is an expressionvector for the expression of a nicotinic acid GPCR in a recombinant hostcell wherein said expression vector comprises, consists essentially of,or consists of the polynucleotide of the twentieth aspect.

Although a variety of expression vectors are available to those in theart, for purposes of utilization for both the endogenous andnon-endogenous human, mouse and rat GPCRs, it is most preferred that thevector utilized be pCMV. In some alternative embodiments as relates tosaid human, mouse and rat nicotinic acid GPCRs, it is preferred that thevector utilized be an adenoviral expression vector.

In a twenty-fourth aspect, the invention features a prokaryotic oreukaryotic host cell comprising, consisting essentially of, orconsisting of the recombinant vector of the twenty-third aspect. In somepreferred embodiments, said host cell is a eukaryotic embryonic stemcell wherein said vector of the twenty-third aspect has been used in amethod to inactivate a gene encoding a nicotinic acid GPCR of theinvention within said cell. In some other preferred embodiments, saidhost cell is a eukaryotic embryonic somatic cell wherein said vector ofthe twenty-third aspect has been used in a method to inactivate a geneencoding a nicotinic acid GPCR of the invention within said cell. Inother preferred embodiments, said host cell is prokaryotic and has beentransformed using the vector of the twenty-third aspect. In furtherpreferred embodiments, said host cell is eukaryotic and has beentransiently transfected using the vector of the twenty-third aspect. Inother further preferred embodiments, said host cell is eukaryotic andhas been stably transfected using the vector of the twenty-third aspect.

In particularly preferred embodiment, said host cell expresses arecombinant nicotinic acid GPCR wherein said host cell comprises,consists essentially of, or consists of the expression vector of thetwenty-third aspect.

A further embodiment includes a prokaryotic or eukaryotic host cellrecombinant for the polynucleotide of the twentieth aspect.

In some embodiments the host cell is eukaryotic, more preferably,mammalian, and more preferably selected from the group consisting of293, 293T, CHO, and COS-7 cells. In other embodiments, the host cell iseukaryotic, more preferably melanophore.

In a twenty-fifth aspect, the invention features a process for theexpression of a nicotinic acid GPCR in a recombinant host cellcomprising the steps of:

(a) transfecting the expression vector of the twenty-third aspect into asuitable host cell; and

(b) culturing the host cells under conditions which allow expression ofthe nicotinic acid GPCR protein from the expression vectors.

In a twenty-sixth aspect, the invention features an antibody thatspecifically binds to the polypeptide of the twenty-first aspect. Insome preferred embodiments, the antibody is monoclonal. In someembodiments, the antibody is polyclonal.

In a twenty-seventh aspect, the invention features a method of bindingthe polypeptide of the twenty-first aspect to the antibody of thetwenty-sixth aspect, comprising contacting said antibody with saidpolypeptide under conditions in which said antibody can specificallybind to said polypeptide.

In a twenty-eighth aspect, the invention features a method of detectinga nicotinic acid GPCR polypeptide in a biological sample obtained froman individual comprising the steps of:

-   -   (a) obtaining said biological sample from said individual;    -   (b) contacting said biological sample with the antibody of the        twenty-sixth aspect; and    -   (c) detecting the presence or absence of binding of said        antibody to said biological sample;        wherein a detection of said binding is indicative of the        receptor polypeptide being expressed in said biological sample.

In preferred embodiments, said detecting is through the use of anenzyme-labeled secondary reagent. In other preferred embodiments, saiddetecting is through the use of a fluorophore-labeled secondary reagent.In other preferred embodiments, said detecting is through the use of aradioisotope-labeled secondary reagent. In other embodiments, theantibody is directly labeled with enzyme, fluorophore or radioisotope.

In other preferred embodiments, said biological sample is taken fromadipose, skin or blood.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In further embodiments, said individual has a disorder of lipidmetabolism selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In further embodiments, said individual has a metabolic-related disorderselected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said method further comprises the step ofcomparing the level of detection of said binding for a first individualto the level of detection of said binding for a second individual.

In a twenty-ninth aspect, the invention features a method of detectingexpression of a gene encoding a nicotinic acid GPCR in a biologicalsample obtained from an individual comprising the steps of:

-   -   (a) obtaining said biological sample from said individual;    -   (b) contacting said biological sample with the complementary        polynucleotide of the twentieth aspect, optionally labeled,        under conditions permissive for hybridization; and    -   (c) detecting the presence or absence of said hybridization        between said complementary polynucleotide and an RNA species        within said sample;        wherein a detection of said hybridization is indicative of        expression of said GPCR gene in said biological sample.

In preferred embodiments, the biological sample is taken from adipose,skin or blood.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In preferred embodiments, said individual has a disorder of lipidmetabolism selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In other preferred embodiments, said individual has a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said method further comprises the step ofcomparing the level of detection of said hybridization for a firstindividual to the level of detection of said hybridization for a secondindividual.

In some preferred embodiments, said complementary polynucleotide is aprimer and said hybridization is detected by detecting the presence ofan amplification product comprising the sequence of said primer. In morepreferred embodiments, said method is RT-PCR.

In a thirtieth aspect, the invention features a GPCR Fusion Proteinconstruct comprising a constitutively active GPCR and a G protein, saidreceptor comprising an amino acid sequence selected from the groupconsisting of:

(a) SEQ. ID. NO.:36 (hRUP25);

(b) SEQ. ID. NO.:137 (mRUP25); and

(c) SEQ. ID. NO.:139 (rRUP25);

or an allelic variant or a biologically active fragment of said aminoacid sequence.

The invention also relates to a GPCR Fusion Protein construct whereinthe isoleucine at amino acid position 230 of SEQ. ID. NO.:36 issubstituted by lysine.

In a thirty-first aspect, the invention features a method of binding aknown ligand of RUP25 nicotinic acid GPCR to a polypeptide selected fromthe group consisting of:

(a) a polypeptide comprising a contiguous span of at least 6 amino acidsof SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(b) a polypeptide comprising a contiguous span of at least 10 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(c) a polypeptide comprising a contiguous span of at least 15 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(d) a polypeptide comprising a contiguous span of at least 20 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(e) a polypeptide comprising a contiguous span of at least 25 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(f) a polypeptide comprising a contiguous span of at least 30 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(g) a polypeptide comprising a contiguous span of at least 35 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(h) a polypeptide comprising a contiguous span of at least 40 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

(i) a polypeptide comprising a contiguous span of at least 45 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139; and

(j) a polypeptide comprising a contiguous span of at least 50 aminoacids of SEQ. ID. NO.:36, SEQ. ID. NO.:137 or SEQ. ID. NO.:139;

or an allelic variant of said polypeptide;

comprising the step of contacting said known ligand with saidpolypeptide under conditions which allow said binding to occur.

In some embodiments, said known ligand is a modulator of the nicotinicacid GPCR. In some embodiments, said known ligand is an agonist of thenicotinic acid GPCR. In other embodiments, said agonist is nicotinicacid or an analog or derivative thereof. In other embodiments, saidagonist is (−)-nicotine or an analog or derivative thereof. In someembodiments, said known ligand is the modulator of the second aspect. Insome embodiments, said known ligand is an antibody specific for theGPCR, or a derivative thereof.

In other preferred embodiments, said method is used to identify whethera candidate compound inhibits said binding of said known ligand to saidpolypeptide, comprising the steps of:

-   -   (a) contacting said polypeptide with said known ligand,        optionally labeled, in the presence or absence of said candidate        compound;    -   (b) detecting the complex between said known ligand and said        polypeptide; and    -   (c) determining whether less of said complex is formed in the        presence of the compound than in the absence of the compound;        wherein said determination is indicative of the candidate        compound being an inhibitor of said binding of said known ligand        to said polypeptide.

In some embodiments, said known ligand is a known modulator of thenicotinic acid GPCR. In some embodiments, said known ligand is amodulator of the nicotinic acid GPCR. In some embodiments, said knownligand is an agonist of the nicotinic acid GPCR. In embodiments, saidagonist is nicotinic acid or an analog or derivative thereof. In otherembodiments, said agonist is (−)-nicotine or an analog or derivativethereof. In some embodiments, said known ligand is the modulator of thesecond aspect. In some embodiments, said known ligand is an antibodyspecific for the GPCR, or a derivative thereof.

In other preferred embodiments, said method is used to identify whethera candidate compound is an inhibitor of said binding of said knownligand to said polypeptide, comprising the steps of:

(a) contacting said polypeptide with said known ligand, optionallylabeled, in the presence separately of a plurality of concentrations ofsaid candidate compound for a time sufficient to allow equilibration ofbinding;

(b) measuring unbound ligand and bound ligand; and

(c) determining K_(i) for the candidate compound;

wherein a K_(i) value of less than 50 μM is indicative of the candidatecompound being an inhibitor of said binding of said known ligand to saidpolypeptide. Preferably said K_(i) value is less than 25 μM, 10 μM, 5μM, 1 μM, 750 nM, 500 nM, 400 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM or 10 nM. Inpreferred embodiments, K_(i) determination is made through nonlinearcurve fitting with the program SCTFIT [De Lean et al. (1982) MolPharmacol 21:5-16; cited in Lorenzen et al. (2001) Mol Pharmacol59:349-357, the disclosures of which are incorporated by referenceherein in their entireties].

In some embodiments, said known ligand is a modulator of the nicotinicacid GPCR. In some embodiments, said known ligand is an agonist of thenicotinic acid GPCR. In other embodiments, said agonist is nicotinicacid or an analog or derivative thereof. In other embodiments, saidagonist is (−)-nicotine or an analog or derivative thereof. In someembodiments, said known ligand is the modulator of the second aspect. Insome embodiments, said known ligand is an antibody specific for theGPCR, or a derivative thereof.

In a thirty-second aspect, the invention features a method of binding anoptionally labeled affinity reagent specific for a nicotinic acid GPCRto said receptor in a biological sample, said receptor comprising anamino acid sequence selected from the group consisting of:

(a) SEQ. ID. NO.:36 (hRUP25);

(b) SEQ. ID. NO.:137 (mRUP25); and

(c) SEQ. ID. NO.:139 (rRUP25);

or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence, comprising the steps of:

(a′) obtaining said biological sample;

(b′) contacting the affinity reagent with said receptor in saidbiological sample; and

(c′) detecting the complex of said affinity reagent with said receptor.

In some embodiments, the nicotinic acid GPCR has an amino acid sequenceselected from the group consisting of:

(a) SEQ. ID. NO.:36 (hRUP25);

(b) SEQ. ID. NO.:137 (mRUP25); and

(c) SEQ. ID. NO.:139 (rRUP25);

-   -   or an allelic variant, a biologically active mutant, or a        biologically active fragment of said amino acid sequence.

In some embodiments, the nicotinic acid GPCR comprises an activefragment of said amino acid sequence.

In some embodiments, the nicotinic acid GPCR is endogenous.

In some embodiments, the nicotinic acid GPCR is recombinant.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:36 further substituted at amino acid position 230 withlysine in place of isoleucine. In preferred embodiments, said EFA mutanthas the amino acid sequence of SEQ. ID. NO.:159.

In preferred embodiments, said G protein is Gi.

In some embodiments, said affinity reagent is a modulator of the GPCR.In some embodiments, said affinity reagent is an agonist of the GPCR. Insome embodiments, said affinity reagent is nicotinic acid or an analogor derivative thereof. In some embodiments, said affinity reagent is(−)-nicotine or an analog or derivative thereof. In some embodiments,said affinity reagent is the modulator of the second aspect. In someembodiments, said known ligand is an antibody specific for the GPCR, ora derivative thereof.

In further preferred embodiments, said affinity reagent comprises alabel selected from the group consisting of:

(a) radioisotope;

(b) enzyme; and

(c) fluorophore.

In preferred embodiments, said radioisotope is ³H.

In a thirty-third aspect, the invention features the method of thethirty-second aspect further comprising the step of comparing the levelof detection of said complex in a first biological sample to a secondlevel of detection of said complex in a second biological sample.

In a thirty-fourth aspect, the invention features the method of thethirty-third aspect wherein the relationship between said first andsecond biological samples is selected from the group consisting of:

(a) said second biological sample is a replicate of said firstbiological sample;

(b) said first biological sample was obtained prior to an experimentalintervention whereas said second biological sample was obtained afterthe experimental intervention, from the same individual;

(c) said second biological sample was obtained at a different time pointafter an experimental intervention than was said first biologicalsample, from the same individual;

(d) said second biological sample corresponds to a different subcellularcompartment than does said first biological sample;

(e) said second biological sample represents a different cell type thandoes said first biological sample;

(f) said second biological sample corresponds to a different tissue thandoes said first biological sample;

(g) said second biological sample was obtained from a differentindividual than was said first biological sample;

(h) said second biological sample was obtained at a different point intime than was said first biological sample, from the same individual;

(i) said first biological samples was obtained from a normal individual,whereas said second biological sample was obtained from an individualhaving a metabolic-related disorder;

(j) said first biological sample was obtained from a normal individual,whereas said second biological sample was obtained from an individualhaving a disorder in lipid metabolism;

(k) said first biological sample was obtained before a therapeuticintervention whereas said second biological sample was obtained afterthe therapeutic intervention, from the same individual;

(l) said second biological sample was obtained at a different time pointafter therapeutic intervention than was said first biological sample,from the same individual; and

(m) said first biological sample was not exposed to a compound, whereassaid second biological sample was exposed to said compound.

In a thirty-fifth aspect, the invention features a method of identifyingwhether a candidate compound is a modulator of an antilipolytic GPCR,said receptor comprising the amino acid sequence of SEQ. ID. NO.:135(hRUP38);

or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence;

comprising the steps of:

(a) contacting the candidate compound with the receptor;

(b) determining whether the receptor functionality is modulated;

wherein a change in receptor functionality is indicative of thecandidate compound being a modulator of an antilipolytic GPCR.

In some embodiments, said antilipolytic GPCR is endogenous.

In some preferred embodiments, said antilipolytic GPCR is recombinant.

Preferred said identified modulator binds to said GPCR.

In some embodiments, said contacting is carried out in the presence of aknown ligand of the GPCR. In some embodiments, said known ligand is anagonist of the GPCR.

The invention also relates to a method of identifying whether acandidate compound is a modulator of lipolysis, comprising the steps of:

-   -   (a) contacting the candidate compound with a GPCR comprising the        amino acid sequence of SEQ. ID. NO.:135 (hRUP38); or an allelic        variant, a biologically active mutant, or a biologically active        fragment of said amino acid sequence; and    -   (b) determining whether the receptor functionality is modulated;        wherein a change in receptor functionality is indicative of the        candidate compound being a modulator of lipolysis.

In some embodiments, said GPCR is endogenous.

In some preferred embodiments, said GPCR is recombinant.

Preferred said identified modulator binds to said GPCR.

In some embodiments, said contacting is carried out in the presence of aknown ligand of the GPCR. In some embodiments, said known ligand is anagonist of the GPCR.

The invention also relates to a method of determining whether acandidate compound is a modulator of an antilipolytic GPCR, comprisingthe steps of:

-   -   (a) culturing antilipolytic GPCR-expressing host cells under        conditions that would allow expression of a recombinant        antilipolytic GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant antilipolytic GPCR        comprising the amino acid sequence of SEQ. ID. NO.:135 (hRUP38);        or an allelic variant, a biologically active mutant, or a        biologically active fragment of said amino acid sequence;    -   (b) contacting the antilipolytic GPCR-expressing host cells of        step (a) with the candidate compound;    -   (c) contacting control host cells with the candidate compound of        step (b), wherein said control host cells do not express        recombinant antilipolytic GPCR protein;    -   (d) measuring the modulating effect of the candidate compound        which interacts with the recombinant antilipolytic GPCR from the        host cells of step (a) and control host cells of step (c); and    -   (e) comparing the modulating effect of the test compound on the        host cells and control host cells.

The invention also relates to a method of determining whether acandidate compound is a modulator of an antilipolytic GPCR, comprisingthe steps of:

-   -   (a) culturing antilipolytic GPCR-expressing host cells under        conditions that would allow expression of a recombinant        antilipolytic GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant antilipolytic GPCR        comprising the amino acid sequence of SEQ. ID. NO.:135 (hRUP38);        or an allelic variant, a biologically active mutant, or a        biologically active fragment of said amino acid sequence;    -   (b) contacting a first population of antilipolytic        GPCR-expressing cells of step (a) with a known ligand of said        antilipolytic GPCR;    -   (c) contacting a second population of antilipolytic        GPCR-expressing cells of step (a) with the candidate compound        and with the known antilipolytic GPCR ligand;    -   (d) contacting control host cells with the candidate compound of        step (c), wherein said control host cells do not express        recombinant antilipolytic GPCR protein;    -   (e) measuring the modulating effect of the candidate compound,        which interacts with recombinant antilipolytic GPCR, in the        presence and absence of the known anti lipolytic GPCR ligand,        from the cells of step (b), step (c) and step (d); and    -   (f) comparing the modulating effect of the candidate compound as        determined from step (b), step (c) and step (d).

In some embodiments, said known ligand is an agonist of the GPCR.

The invention also relates to a method of determining whether acandidate compound is a modulator of an antilipolytic GPCR, comprisingthe steps of:

-   -   (a) culturing antilipolytic GPCR-expressing host cells under        conditions that would allow expression of a recombinant        antilipolytic GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant antilipolytic GPCR        comprising the amino acid sequence of SEQ. ID. NO.:135 (hRUP38);        or an allelic variant, a biologically active mutant, or a        biologically active fragment of said amino acid sequence;    -   (b) contacting a first population of the antilipolytic        GPCR-expressing host cells of step (a) with the candidate        compound;    -   (c) not contacting a second population of the antilipolytic        GPCR-expressing cells of step (a) with the candidate compound of        step (b);    -   (d) contacting control host cells to the candidate compound of        step (b), wherein said control host cells do not express        recombinant antilipolytic GPCR protein;    -   (e) measuring the modulating effect of the candidate compound,        which interacts with recombinant antilipolytic GPCR protein,        from the cells of step (b) and step (c) and from the cells of        step (d); and    -   (f) comparing the modulating effect of the candidate compound as        determined from step (b) and step (c) and from step (d).

In some embodiments, the antilipolytic GPCR has the amino acid sequenceof SEQ. ID. NO.:135 (hRUP38); or an allelic variant, a biologicallyactive mutant, or a biologically active fragment of said amino acidsequence.

In some embodiments, the antilipolytic GPCR comprises a biologicallyactive fragment of said amino acid sequence.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:135 further substituted at amino acid position 230 withlysine in place of isoleucine.

In preferred embodiments, said G protein is Gi.

In other preferred embodiments, said determining is through the use of aMelanophore assay.

In other preferred embodiments, said determining is through themeasurement of the level of a second messenger selected from the groupconsisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositoltriphosphate (IP₃), diacylglycerol (DAG), and Ca²⁺. In further preferredembodiments, said second messenger is cAMP. In more preferredembodiments, the level of the cAMP is reduced. In some embodiments, saidmeasurement of cAMP is carried out with membrane comprising said GPCR.

In other preferred embodiments, said determining is through themeasurement of an activity up-regulated or down-regulated by a reductionin intracellular cAMP level. In further preferred embodiments, saiddown-regulated activity is intracellular lipolysis. In other furtherpreferred embodiments, said down-regulated activity is hormone sensitivelipase activity. In other further preferred embodiments, saidup-regulated activity is adiponectin secretion.

In other preferred embodiments, said determining is through CRE-reporterassay. In preferred embodiments, said reporter is luciferase. In someembodiments, said reporter is β-galactosidase.

In other embodiments, said recombinant host cell further comprisespromiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit and saiddetermining is through measurement of intracellular Ca²⁺. In preferredembodiments, said Ca²⁺ measurement is carried out by FLIPR.

In other embodiments, said recombinant host cell further comprisespromiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit and saiddetermining is through measurement of intracellular IP₃.

In other preferred embodiments, said determining is through themeasurement of GTPγS binding to membrane comprising said GPCR. Infurther preferred embodiments, said GTPγS is labeled with [³⁵S].

In other preferred embodiments, said method further comprises the stepof comparing the modulation of the receptor caused by the candidatecompound to a second modulation of the receptor caused by contacting thereceptor with a known modulator of the receptor. In some preferredembodiments, said known modulator is an agonist.

In a thirty-sixth aspect, the invention features a modulator of anantilipolytic GPCR identified according to the method of thethirty-fifth aspect.

In some preferred embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.More preferably, said modulator is an agonist. In some embodiments, saidmodulator is a partial agonist.

In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 1000 μM in GTPγS binding assay carried out with membrane fromstably transfected CHO cells expressing recombinant hRUP38 polypeptidehaving the amino acid sequence of SEQ. ID. NO.:135. In some embodiments,said modulator is an agonist with an EC₅₀ of less than 900 μM in saidassay. In some embodiments, said modulator is an agonist with an EC₅₀ ofless than 800 μM in said assay. In some embodiments, said modulator isan agonist with an EC₅₀ of less than 700 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 600μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 550 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 500 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 450 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 400 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 350μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 300 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 250 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 200 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 150 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 100μM in said assay. In some preferred embodiments, said modulator is anagonist with an EC₅₀ in said assay of less than a value selected fromthe interval of 600 μM to 1000 μM.

In some embodiments, said modulator is selective for the GPCR.

In some embodiments, said modulator is antilipolytic.

In some embodiments, said modulator is orally bioavailable. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some preferred embodiments, said oral bioavailabilityis at least 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In some preferredembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In highly less preferred embodiments, said modulator is an antibody orderivative thereof.

In a thirty-seventh aspect, the invention features the method of thethirty-fifth aspect, wherein said candidate compound is an agonist ofhRUP25 GPCR comprising the amino acid sequence of SEQ. ID. NO.:36 andwherein said method further comprises the step of comparing themodulation of hRUP38 GPCR comprising the amino acid sequence of SEQ. ID.NO.:135 caused by said agonist to a second modulation of hRUP38 GPCRcomprising a variant of said amino acid sequence caused by contactingthe variant hRUP38 GPCR with said agonist.

In preferred embodiments, said variant amino acid sequence is identicalto hRUP38 polypeptide of SEQ. ID. NO.:135, further comprising a singleamino acid substitution selected from the group consisting of:

(a) V for A at amino acid position 27 of SEQ. ID. NO.:135;

(b) L for V at amino acid position 83 of SEQ. ID. NO.:135;

(c) N for Y at amino acid position 86 of SEQ. ID. NO.:135;

(d) W for S at amino acid position 91 of SEQ. ID. NO.:135;

(e) K for N at amino acid position 94 of SEQ. ID. NO.:135;

(f) M for V at amino acid position 103 of SEQ. ID. NO.:135;

(g) L for F at amino acid position 107 of SEQ. ID. NO.:135;

(h) R for W at amino acid position 142 of SEQ. ID. NO.:135;

(i) I for V at amino acid position 156 of SEQ. ID. NO.:135;

(j) M for L at amino acid position 167 of SEQ. ID. NO.:135;

(k) P for L at amino acid position 168 of SEQ. ID. NO.:135;

(l) G for P at amino acid position 173 of SEQ. ID. NO.:135;

(m) L for V at amino acid position 176 of SEQ. ID. NO.:135;

(n) S for 1 at amino acid position 178 of SEQ. ID. NO.:135;

(O) Q for Rat amino acid position 187 of SEQ. ID. NO.:135;

(p) F for L at amino acid position 198 of SEQ. ID. NO.:135; and

(q) P for N at amino acid position 363 of SEQ. ID. NO.:135.

In particularly preferred embodiments, said method is used to identifywhether said substituted amino acid additionally found at the identicalposition within SEQ. ID. NO.:36 is necessary for modulation of saidhRUP25 GPCR by said agonist, comprising the steps of:

(a) determining the level of modulation of said hRUP38 GPCR by saidagonist; and

(b) determining the level of modulation of said variant hRUP38 GPCR bysaid agonist;

wherein if said level of modulation for (b) is greater than said levelof modulation for (a), then said substituted amino acid is necessary formodulation of said hRUP25 GPCR by said agonist.

In a thirty-eighth aspect, the invention features a method of modulatingthe activity of an antilipolytic GPCR, said receptor comprising theamino acid sequence of SEQ. ID. NO.:135 (hRUP38); or an allelic variant,a biologically active mutant, or a biologically active fragment of saidamino acid sequence; comprising the step of contacting the receptor withthe modulator of the thirty-sixth aspect.

In some embodiments, the antilipolytic GPCR has the amino acid sequenceof SEQ. ID. NO.:135 (hRUP38); or an allelic variant, a biologicallyactive mutant, or a biologically active fragment of said amino acidsequence.

In some embodiments, the antilipolytic GPCR comprises an active fragmentof said amino acid sequence.

In some embodiments, the antilipolytic GPCR is endogenous.

In some embodiments, the antilipolytic GPCR is recombinant.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:135 further substituted at amino acid position 230 withlysine in place of isoleucine.

In preferred embodiments, said G protein is Gi.

In some preferred embodiments, said modulator is an agonist.

In preferred embodiments, said modulator is selective for the GPCR.

In other preferred embodiments, said contacting comprises administrationof the modulator to a membrane comprising the receptor.

In other preferred embodiments, said contacting comprises administrationof the modulator to a cell or tissue comprising the receptor.

In other preferred embodiments, said contacting comprises administrationof the modulator to an individual comprising the receptor. In morepreferred embodiments, said individual is a mammal. In other morepreferred embodiments, said mammal is a horse, cow, sheep, pig, cat,dog, rabbit, mouse, rat, non-human primate or human. Yet more preferredis mouse, rat or human. Most preferred is human.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP38polypeptide having the amino acid sequence of SEQ. ID. NO.:135. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said administration is oral.

In preferred embodiments, said modulator is an agonist and saidindividual is in need of prevention of or treatment for ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said modulator is an inverse agonist and saidmetabolic-related disorder relates to a low level of plasma free fattyacids.

In other preferred embodiments, said modulator is an agonist and saidindividual is in need of a change in lipid metabolism selected from thegroup consisting of:

(a) a decrease in the level of plasma triglycerides;

(b) a decrease in the level of plasma free fatty acids;

(c) a decrease in the level of plasma cholesterol;

(d) a decrease in the level of LDL-cholesterol;

(e) an increase in the level of HDL-cholesterol;

(f) a decrease in the total cholesterol/HDL-cholesterol ratio; and

(g) an increase in the level of plasma adiponectin.

In other preferred embodiments, said needed change in lipid metabolismis a decrease in the postprandial increase in plasma free fatty acidsdue to a high fat meal or an inhibition of the progression from impairedglucose tolerance to insulin resistance.

In some embodiments, the modulator is an inverse agonist and the neededchange in lipid metabolism is an increase in the level of plasma freefatty acids.

In other preferred embodiments, said modulator is an agonist and saidindividual is a mouse genetically predisposed to a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure.

In further preferred embodiments, said metabolic-related disorder ishyperlipidemia.

In further preferred embodiments, said method is used to identifywhether said agonist has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering said agonist to the mouse; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering said agonist compared to not administeringsaid agonist;

wherein said determination is indicative of said agonist havingtherapeutic efficacy.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other preferred embodiments, said modulator is an agonist and saidindividual is a rat genetically predisposed to a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Infurther preferred embodiments, said metabolic-related disorder ishyperlipidemia.

In further preferred embodiments, said method is used to identifywhether said agonist has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering said agonist to the rat; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering said agonist compared to not administeringsaid agonist; wherein said determination is indicative of said agonisthaving therapeutic efficacy.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In a thirty-ninth aspect, the invention features a method of preventingor treating a disorder of lipid metabolism in an individual comprisingcontacting a therapeutically effective amount of the modulator of thethirty-sixth aspect with an antilipolytic GPCR, said receptor comprisingthe amino acid sequence of SEQ. ID. NO.:135 (hRUP38); or an allelicvariant or biologically active fragment of said amino acid sequence.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP38polypeptide having the amino acid sequence of SEQ. ID. NO.:135. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said contacting comprises oraladministration of said modulator to said individual.

In preferred embodiment, said modulator is an agonist and said disorderof lipid metabolism is selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In a fortieth aspect, the invention features a method of preventing ortreating a metabolic-related disorder in an individual comprisingcontacting a therapeutically effective amount of the modulator of thethirty-sixth aspect with an antilipolytic GPCR, said receptor comprisingthe amino acid sequence of SEQ. ID. NO.:135 (hRUP38); or an allelicvariant or biologically active fragment of said amino acid sequence.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀Of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP38polypeptide having the amino acid sequence of SEQ. ID. NO.:135. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said contacting comprises oraladministration of said modulator to said individual.

In preferred embodiment, said modulator is an agonist and saidmetabolic-related disorder is selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In a forty-first aspect, the invention features a method of preparing acomposition which comprises identifying a modulator of an antilipolyticGPCR and then admixing a carrier and the modulator, wherein themodulator is identifiable by the method of the thirty-fifth aspect.

In some preferred embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.More preferably, said modulator is an agonist. In some embodiments, saidmodulator is a partial agonist.

In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 1000 μM in GTPγS binding assay carried out with membrane fromstably transfected CHO cells expressing recombinant hRUP38 polypeptidehaving the amino acid sequence of SEQ. ID. NO.:135. In some embodiments,said modulator is an agonist with an EC₅₀ of less than 900 μM in saidassay. In some embodiments, said modulator is an agonist with an EC₅₀ ofless than 800 μM in said assay. In some embodiments, said modulator isan agonist with an EC₅₀ of less than 700 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 600μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 550 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 500 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 450 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 400 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 350μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 300 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 250 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 200 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 150 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 100μM in said assay. In some preferred embodiments, said modulator is anagonist with an EC₅₀ in said assay of less than a value selected fromthe interval of 600 μM to 1000 μM.

In some embodiments, said modulator is selective for the GPCR.

In some embodiments, said modulator is antilipolytic.

In some embodiments, said modulator is orally bioavailable. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to eitherintraperitoneal or intravenous administration. In some preferredembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to either intraperitoneal orintravenous administration.

In a forty-second aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of the modulator of the thirty-sixthaspect. In preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP38polypeptide having the amino acid sequence of SEQ. ID. NO.:135. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In a forty-third aspect, the invention features a method of changinglipid metabolism comprising providing or administering to an individualin need of said change said pharmaceutical or physiologically acceptablecomposition of the forty-second aspect, said needed change in lipidmetabolism selected from the group consisting of:

(a) a decrease in the level of plasma triglycerides;

(b) a decrease in the level of plasma free fatty acids;

(c) a decrease in the level of plasma cholesterol;

(d) a decrease in the level of LDL-cholesterol;

(e) an increase in the level of HDL-cholesterol;

(f) a decrease in the total cholesterol/HDL-cholesterol ratio; and

(g) an increase in the level of plasma adiponectin.

In preferred embodiments, a therapeutically effective amount of saidpharmaceutical or physiologically acceptable composition is provided oradministered to said individual.

In some preferred embodiments, said providing or administering of saidpharmaceutical or physiologically acceptable composition is oral.

In other preferred embodiments, said needed change in lipid metabolismis a decrease in the postprandial increase in plasma free fatty acidsdue to a high fat meal or an inhibition of the progression from impairedglucose tolerance to insulin resistance.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In a forty-fourth aspect, the invention features a method of preventingor treating a metabolic-related disorder comprising providing oradministering to an individual in need of said treatment saidpharmaceutical or physiologically acceptable composition of theforty-second aspect, said metabolic-related disorder selected from thegroup consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In preferred embodiments, a therapeutically effective amount of saidpharmaceutical or physiologically acceptable composition is provided oradministered to said individual.

In some preferred embodiments, said providing or administering of saidpharmaceutical or physiologically acceptable composition is oral.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, stroke, Syndrome X, andheart disease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In an forty-fifth aspect, the invention features a method of using themodulator of the thirty-sixth aspect for the preparation of a medicamentfor the treatment of a disorder in lipid metabolism in an individual.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP38polypeptide having the amino acid sequence of SEQ. ID. NO.:135. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said treatment comprises oraladministration of said medicament to said individual.

In preferred embodiments, said modulator is an agonist and said disorderin lipid metabolism is selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In a forty-sixth aspect, the invention features a method of using themodulator of the thirty-sixth aspect for the preparation of a medicamentfor the treatment of a metabolic-related disorder in an individual.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP38polypeptide having the amino acid sequence of SEQ. ID. NO.:135. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said treatment comprises oraladministration of said medicament to said individual.

In preferred embodiments, said modulator is an agonist and saidmetabolic-related disorder is selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In a forty-seventh aspect, the invention features a method ofidentifying whether a candidate compound is binds to an antilipolyticGPCR, said receptor comprising the amino acid sequence of SEQ. ID.NO.:135 (hRUP38); or an allelic variant or a biologically activefragment of said amino acid sequence; comprising the steps of:

-   -   (a) contacting the receptor with a labeled reference compound        known to bind to the GPCR in the presence or absence of the        candidate compound; and    -   (b) determining whether the binding of said labeled reference        compound to the receptor is inhibited in the presence of the        candidate compound; wherein said inhibition is indicative of the        candidate compound binding to an antilipolytic GPCR.

In some embodiments, the antilipolytic GPCR comprises a biologicallyactive fragment of said amino acid sequence.

In some embodiments, the antilipolytic GPCR is endogenous.

In some embodiments, the antilipolytic GPCR is recombinant.

In preferred embodiments, said G protein is Gi.

In some preferred embodiments, said reference compound is the modulatorof the thirty-sixth aspect.

In other embodiments, said reference compound is an antibody specificfor the GPCR, or a derivative thereof.

In preferred embodiments, said reference compound comprises a labelselected from the group consisting of:

(a) radioisotope;

(b) enzyme; and

(c) fluorophore.

In some preferred embodiments, said label is ³H.

In other embodiments, said method further comprises the step ofcomparing the level of inhibition of binding of a labeled firstreference compound by the candidate compound to a second level ofinhibition of binding of said labeled first reference compound by asecond reference compound known to bind to the GPCR.

In a forty-eighth aspect, the invention features a method of making atransgenic mouse, comprising the step of engineering said mouse to carryas part of its own genetic material the gene encoding the humanantilipolytic GPCR polypeptide of SEQ. ID. NO.:135 (hRUP38).

In some preferred embodiments, expression of said gene is placed underthe control of an essentially adipocyte specific promoter.

In a forty-ninth aspect, the invention features the transgenic mouseaccording to the method of the forty-eighth aspect.

In a fiftieth aspect, the invention features a method of using thetransgenic mouse of the forty-ninth aspect to identify whether anagonist of said human receptor has therapeutic efficacy for thetreatment of a disorder of lipid metabolism selected from the groupconsisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin; comprising the steps of:

(a′) administering or not administering the agonist to the mouse; and

(b′) determining whether on administering the agonist there is a changeselected from the group consisting of:

-   -   (i) a decrease in the level of plasma triglycerides;    -   (ii) a decrease in the level of plasma free fatty acids;    -   (iii) a decrease in the level of plasma cholesterol;    -   (iv) a decrease in the level of LDL-cholesterol;    -   (v) an increase in the level of HDL-cholesterol;    -   (vi) a decrease in the total cholesterol/HDL-cholesterol ratio;        and    -   (vii) an increase in the level of plasma adiponectin;        wherein said change is indicative of the agonist having        therapeutic efficacy.

In a fifty-first aspect, the invention features a method of using thetransgenic mouse of the forty-ninth aspect to identify whether anagonist of said human receptor has therapeutic efficacy for thetreatment of a metabolic-related disorder selected from the groupconsisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering the agonist to the mouse; and

(b′) determining whether on administering the agonist there is a changeselected from the group consisting of:

-   -   (i) a decrease in the level of plasma triglycerides;    -   (ii) a decrease in the level of plasma free fatty acids;    -   (iii) a decrease in the level of plasma cholesterol;    -   (iv) a decrease in the level of LDL-cholesterol;    -   (v) an increase in the level of HDL-cholesterol;    -   (vi) a decrease in the total cholesterol/HDL-cholesterol ratio;        and    -   (vii) an increase in the level of plasma adiponectin;        wherein said change is indicative of the agonist having        therapeutic efficacy.

In a fifty-second aspect, the invention features a method of making atransgenic rat, comprising the step of engineering said rat to carry aspart of its own genetic material the gene encoding the humanantilipolytic GPCR polypeptide of SEQ. ID. NO.:135 (hRUP38).

In some preferred embodiments, expression of said gene is placed underthe control of an essentially adipocyte specific promoter.

In a fifty-third aspect, the invention features the transgenic rataccording to the method of the fifty-second aspect.

In a fifty-fourth aspect, the invention features a method of using thetransgenic rat of the fifty-third aspect to identify whether an agonistof said human receptor has therapeutic efficacy for the treatment of adisorder of lipid metabolism selected from the group consisting of:

-   -   (a) elevated level of plasma triglycerides;    -   (b) elevated level of plasma free fatty acids;    -   (c) elevated level of plasma cholesterol;    -   (d) elevated level of LDL-cholesterol;    -   (e) reduced level of HDL-cholesterol;    -   (f) elevated total cholesterol/HDL-cholesterol ratio; and    -   (g) reduced level of plasma adiponectin;        comprising the steps of:

(a′) administering or not administering the agonist to the rat; and

(b′) determining whether on administering the agonist there is a changeselected from the group consisting of:

-   -   (i) a decrease in the level of plasma triglycerides;    -   (ii) a decrease in the level of plasma free fatty acids;    -   (iii) a decrease in the level of plasma cholesterol;    -   (iv) a decrease in the level of LDL-cholesterol;    -   (v) an increase in the level of HDL-cholesterol;    -   (vi) a decrease in the total cholesterol/HDL-cholesterol ratio;        and    -   (vii) an increase in the level of plasma adiponectin;        wherein said change is indicative of the agonist having        therapeutic efficacy.

In a fifty-fifth aspect, the invention features a method of using thetransgenic rat of the fifty-third aspect to identify whether an agonistof said human receptor has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

-   -   (a) dyslipidemia;    -   (b) atherosclerosis;    -   (c) coronary heart disease;    -   (d) stroke;    -   (e) insulin resistance; and    -   (f) type 2 diabetes;        comprising the steps of:

(a′) administering or not administering the agonist to the rat; and

(b′) determining whether on administering the agonist there is a changeselected from the group consisting of:

-   -   (i) a decrease in the level of plasma triglycerides;    -   (ii) a decrease in the level of plasma free fatty acids;

(iii) a decrease in the level of plasma cholesterol;

-   -   (iv) a decrease in the level of LDL-cholesterol;    -   (v) an increase in the level of HDL-cholesterol;    -   (vi) a decrease in the total cholesterol/HDL-cholesterol ratio;        and    -   (vii) an increase in the level of plasma adiponectin;        wherein said change is indicative of the agonist having        therapeutic efficacy.

In a fifty-sixth aspect, the invention features an isolated, purified orrecombinant RUP38 polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising a contiguous span of at least 75nucleotides of SEQ.ID. NO.:134, or an allelic variant of saidpolynucleotide;

(b) a polynucleotide comprising a contiguous span of at least 150nucleotides of SEQ. ID. NO.:134, or an allelic variant of saidpolynucleotide;

(c) a polynucleotide comprising a contiguous span of at least 250nucleotides of SEQ. ID. NO.:134, or an allelic variant of saidpolynucleotide;

(d) a polynucleotide comprising a contiguous span of at least 350nucleotides of SEQ. ID. NO.:134, or an allelic variant of saidpolynucleotide;

(e) a polynucleotide comprising a contiguous span of at least 500nucleotides of SEQ. ID. NO.:134, or an allelic variant of saidpolynucleotide;

(f) a polynucleotide comprising a contiguous span of at least 750nucleotides of SEQ. ID. NOs.:134, or an allelic variant of saidpolynucleotide;

(g) a polynucleotide comprising a contiguous span of at least 1000nucleotides of SEQ. ID. NO.:134, or an allelic variant of saidpolynucleotide;

(h) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 20 amino acids of SEQ. ID. NO.:135 or an allelic variant ofsaid polypeptide;

(i) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 30 amino acids of SEQ. ID. NO.:135, or an allelic variant ofsaid polypeptide;

(j) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 40 amino acids of SEQ. ID. NO.:135, or an allelic variant ofsaid polypeptide;

(k) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 50 amino acids of SEQ. ID. NO.:135 or an allelic variant ofsaid polypeptide;

(l) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 75 amino acids of SEQ. ID. NO.:135 or an allelic variant ofsaid polypeptide;

(m) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 100 amino acids of SEQ. ID. NO.:135 or an allelic variant ofsaid polypeptide;

(n) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 150 amino acids of SEQ. ID. NO.:135 or an allelic variant ofsaid polypeptide;

(O) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 200 amino acids of SEQ. ID. NO.:135 or an allelic variant ofsaid polypeptide;

(p) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 250 amino acids of SEQ. ID. NO.:135 or an allelic variant ofsaid polypeptide; and

(q) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 300 amino acids of SEQ.ID. NO.:135 or an allelic variant ofsaid polypeptide.

The invention also relates to an isolated, purified or recombinant RUP25polynucleotide wherein said polynucleotide is selected from the groupconsisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ. ID.NO.:134 or an allelic variant of said nucleotide sequence;

(b) the polynucleotide of SEQ. ID. NO.:134, or an allelic variant ofsaid polynucleotide;

(c) a polynucleotide comprising a nucleotide sequence encoding apolypeptide having the amino acid sequence of SEQ. ID. NO.:135 or anallelic variant of said amino acid sequence; and

(d) a polynucleotide encoding a polypeptide having the amino acidsequence of SEQ. ID. NO.:135, or an allelic variant of said polypeptide.

In preferred embodiments, said isolated, purified or recombinantpolynucleotide comprises at least 8 contiguous nucleotides of apolynucleotide of the present invention. In other preferred embodiments,said isolated, purified or recombinant polynucleotide comprises at least10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,500, 600, 700, 800, 900 or 1000 contiguous nucleotides of apolynucleotide of the present invention. Preferably said polynucleotideencodes full-length RUP38 polypeptide or a biologically active fragmentthereof.

The polynucleotides of the present invention include genomicpolynucleotides comprising RUP38 polynucleotides of the invention.

The present invention also relates to a polynucleotide encoding a fusionprotein, wherein said fusion protein comprises an RUP38 polypeptide ofthe invention fused to a heterologous polypeptide. In a preferredembodiment, said polypeptide of the invention is constitutively activeand said heterologous polypeptide is a G protein. In other embodiments,said heterologous polypeptide provides an antigenic epitope. In apreferred embodiment, said heterologous polypeptide provides ahemaglutinin (HA) antigenic epitope. Methods relating to apolynucleotide encoding a fusion protein are well known to those ofordinary skill in the art.

The polynucleotides of the present invention also include variantpolynucleotides at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%identical to an RUP38 polynucleotide of the invention. In a particularlypreferred embodiments, polynucleotide sequence homologies are evaluatedusing the Basic Local Alignment Search Tool (“BLAST”), which is wellknown in the art [See, e.g., Karlin and Altschul, Proc Natl Acad Sci USA(1990) 87:2264-8; Altschul et al., J Mol Biol (1990) 215:403-410;Altschul et al., Nature Genetics (1993) 3:266-72; and Altschul et al.,Nucleic Acids Res (1997) 25:3389-3402; the disclosures of which areincorporated by reference in their entirety].

In further preferred embodiments, the invention features the complementof said polynucleotide.

In a fifty-seventh aspect, the invention features an isolated, purifiedor recombinant RUP38 polypeptide selected from the group consisting of:

(a) a polypeptide comprising a contiguous span of at least 20 aminoacids of SEQ. ID. NO.:135 or an allelic variant of said contiguous spanof amino acids;

(b) a polypeptide comprising a contiguous span of at least 30 aminoacids of SEQ. ID. NO.:135 or an allelic variant of said contiguous spanof amino acids;

(c) a polypeptide comprising a contiguous span of at least 40 aminoacids of SEQ. ID. NO.:135 or an allelic variant of said contiguous spanof amino acids;

(d) a polypeptide comprising a contiguous span of at least 50 aminoacids of SEQ. ID. NO.:135 or an allelic variant of said contiguous spanof amino acids;

(e) a polypeptide comprising a contiguous span of at least 75 aminoacids of SEQ. ID. NO.:135 or an allelic variant of said contiguous spanof amino acids;

(f) a polypeptide comprising a contiguous span of at least 100 aminoacids of SEQ. ID. NO.:135 or an allelic variant of said contiguous spanof amino acids;

(g) a polypeptide comprising a contiguous span of at least 150 aminoacids of SEQ. ID. NO.:135 or an allelic variant of said contiguous spanof amino acids;

(h) a polypeptide comprising a contiguous span of at least 200 aminoacids of SEQ. ID. NO.:135 or an allelic variant of said contiguous spanof amino acids;

(i) a polypeptide comprising a contiguous span of at least 250 aminoacids of SEQ. ID. NO.:135 or an allelic variant of said contiguous spanof amino acids; and

(j) a polypeptide comprising a contiguous span of at least 300 aminoacids of SEQ. ID. NO.:135 or an allelic variant of said contiguous spanof amino acids.

The invention also relates to an isolated, purified or recombinant RUP38polypeptide wherein said polypeptide is selected from the groupconsisting of:

(a) a polypeptide comprising the amino acid sequence of SEQ. ID. NO.:135or an allelic variant or a biologically active mutant of said amino acidsequence; and

(b) the polypeptide having the amino acid sequence of SEQ. ID. NO.:135or an allelic variant or a biologically active mutant of said amino acidsequence; or a biologically active fragment of said polypeptide.

In preferred embodiments, said isolated, purified or recombinantpolypeptide comprises at least 6 contiguous amino acids of an RUP38polypeptide of the invention. In further embodiments, said isolated,purified or recombinant polypeptide comprises at least 10, 12, 15, 20,25, 30, 35, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275 or300 contiguous amino acids of a polypeptide of the present invention.Preferably, said polypeptide is full-length RUP38 polypeptide or anactive fragment thereof.

The present invention also relates to a fusion protein, wherein saidfusion protein comprises an RUP38 polypeptide of the invention fused toa heterologous polypeptide. In a preferred embodiment, said polypeptideof the invention is constitutively active and said heterologouspolypeptide is a G protein. In other preferred embodiments, saidheterologous polypeptide provides an antigenic epitope. In particularlypreferred embodiment, said heterologous polypeptide provides ahemaglutinin (HA) antigenic epitope. Methods relating to a fusionprotein are well known to those of ordinary skill in the art.

The polypeptides of the present invention also include variantpolypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to an RUP38 polypeptide of the invention. In aparticularly preferred embodiments, polypeptide sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”), whichis well known in the art [See, e.g., Karlin and Altschul, Proc Natl AcadSci USA (1990) 87:2264-8; Altschul et al., J Mol Biol (1990) 215:403410;Altschul et al., Nature Genetics (1993) 3:266-72; and Altschul et al.,Nucleic Acids Res (1997) 25:3389-3402; the disclosures of which areincorporated by reference in their entirety].

In an fifty-eighth aspect, the invention features a compositioncomprising, consisting essentially of, or consisting of the RUP38polypeptide of the fifty-seventh aspect.

In a fifty-ninth aspect, the invention features a recombinant vector,said vector comprising, consisting essentially of, or consisting of thepolynucleotide of the fifty-sixth aspect. In some embodiments, saidvector is a targeting vector used in a method of inactivating a geneencoding an antilipolytic GPCR of the invention. In some preferredembodiments, said vector is used in a method of transient or stabletransfection. In other preferred embodiments, said vector is used in amethod of transgenic expression of an antilipolytic GPCR.

In particularly preferred embodiment, said vector is an expressionvector for the expression of a an antilipolytic GPCR in a recombinanthost cell wherein said expression vector comprises, consists essentiallyof, or consists of the polynucleotide of the fifty-sixth aspect.Although a variety of expression vectors are available to those in theart, for purposes of utilization for both the endogenous andnon-endogenous human, mouse and rat GPCRs, it is most preferred that thevector utilized be pCMV. In some alternative embodiments as relates tosaid human, mouse and rat antilipolytic GPCRs, it is preferred that thevector utilized be an adenoviral expression vector.

In a sixtieth aspect, the invention features a prokaryotic or eukaryotichost cell comprising, consisting essentially of, or consisting of therecombinant vector of the fifty-ninth aspect. In some embodiments, saidhost cell is a eukaryotic embryonic stem cell wherein said vector of thefifty-ninth aspect has been used in a method to inactivate a geneencoding an antilipolytic GPCR of the invention within said cell. Insome embodiments, said host cell is a eukaryotic embryonic somatic cellwherein said vector of the fifty-ninth aspect has been used in a methodto inactivate a gene encoding an antilipolytic GPCR of the inventionwithin said cell. In some preferred embodiments, said host cell isderived from a mouse or rat made transgenic for a human RUP38antilipolytic GPCR of the invention. In some preferred embodiments, saidhost cell is prokaryotic and has been transformed using the vector ofthe fifty-ninth aspect. In further preferred embodiments, said host cellis eukaryotic and has been transiently transfected using the vector ofthe fifty-ninth aspect. In other further preferred embodiments, saidhost cell is eukaryotic and has been stably transfected using the vectorof the fifty-ninth aspect.

In particularly preferred embodiment, said host cell expresses arecombinant antilipolytic GPCR wherein said host cell comprises,consists essentially of, or consists of the expression vector of thefifty-ninth aspect.

A further embodiment includes a prokaryotic or eukaryotic host cellrecombinant for the polynucleotide of the fifty-sixth aspect.

In some embodiments the host cell is eukaryotic, more preferably,mammalian, and more preferably selected from the group consisting of293, 293T, CHO, and COS-7 cells. In other embodiments, the host cell iseukaryotic, more preferably melanophore.

In a sixty-first aspect, the invention features a process for theexpression of a antilipolytic GPCR in a recombinant host cell comprisingthe steps of:

-   -   (a) transfecting the expression vector of the fifty-ninth aspect        into a suitable host cell; and    -   (b) culturing the host cells under conditions which allow        expression of the antilipolytic GPCR protein from the expression        vectors.

In a sixty-second aspect, the invention features an antibody thatspecifically binds to the polypeptide of the fifty-seventh aspect. Insome preferred embodiments, the antibody is monoclonal. In someembodiments, the antibody is polyclonal.

In a sixty-third aspect, the invention features a method of binding thepolypeptide of the fifty-seventh aspect to the antibody of thesixty-second aspect, comprising contacting said antibody with saidpolypeptide under conditions in which said antibody can specificallybind to said polypeptide.

In a sixty-fourth aspect, the invention features a method of detectingan antilipolytic GPCR polypeptide in a biological sample obtained froman individual comprising the steps of:

(a) obtaining said biological sample from said individual;

(b) contacting said biological sample with the antibody of thesixty-second aspect; and

(c) detecting the presence or absence of binding of said antibody tosaid biological sample;

wherein a detection of said binding is indicative of the receptorpolypeptide being expressed in said biological sample.

In preferred embodiments, said detecting is through the use of anenzyme-labeled secondary reagent. In other preferred embodiments, saiddetecting is through the use of a fluorophore-labeled secondary reagent.In other preferred embodiments, said detecting is through the use of aradioisotope-labeled secondary reagent. In other embodiments, theantibody is directly labeled with enzyme, fluorophore or radioisotope.

In other preferred embodiments, said biological sample is taken fromadipose, skin or blood.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In further embodiments, said individual has a disorder of lipidmetabolism selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In further embodiments, said individual has a metabolic-related disorderselected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said method further comprises the step ofcomparing the level of detection of said binding for a first individualto the level of detection of said binding for a second individual.

In a sixty-fifth aspect, the invention features a method of detectingexpression of a gene encoding an antilipolytic GPCR in a biologicalsample obtained from an individual comprising the steps of:

-   -   (a) obtaining said biological sample from said individual;    -   (b) contacting said biological sample with the complementary        polynucleotide of the fifty-sixth aspect, optionally labeled,        under conditions permissive for hybridization; and    -   (c) detecting the presence or absence of said hybridization        between said complementary polynucleotide and an RNA species        within said sample;        wherein a detection of said hybridization is indicative of        expression of said GPCR gene in said biological sample.

In preferred embodiments, the biological sample is taken from adipose,skin or blood.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In preferred embodiments, said individual has a disorder of lipidmetabolism selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In other preferred embodiments, said individual has a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said method further comprises the step ofcomparing the level of detection of said hybridization for a firstindividual to the level of detection of said hybridization for a secondindividual.

In some preferred embodiments, said complementary polynucleotide is aprimer and said hybridization is detected by detecting the presence ofan amplification product comprising the sequence of said primer. In morepreferred embodiments, said method is RT-PCR.

In a sixty-sixth aspect, the invention features a GPCR Fusion Proteinconstruct comprising a constitutively active GPCR and a G protein, saidreceptor comprising the amino acid sequence of SEQ. ID. NO.:135 (hRUP38)or an allelic variant or a biologically active fragment of said aminoacid sequence.

The invention also relates to a GPCR Fusion Protein construct whereinthe isoleucine at amino acid position 230 of SEQ. ID. NO.:135 issubstituted by lysine.

In a sixty-seventh aspect, the invention features a method of binding aknown ligand of RUP38 antilipolytic GPCR to a polypeptide selected fromthe group consisting of:

(a) a polypeptide comprising a contiguous span of at least 6 amino acidsof SEQ. ID. NO.:135;

(b) a polypeptide comprising a contiguous span of at least 10 aminoacids of SEQ. ID. NO.:135;

(c) a polypeptide comprising a contiguous span of at least 15 aminoacids of SEQ. ID. NO.:135;

(d) a polypeptide comprising a contiguous span of at least 20 aminoacids of SEQ. ID. NO.:135;

(e) a polypeptide comprising a contiguous span of at least 25 aminoacids of SEQ. ID. NO.:135;

(f) a polypeptide comprising a contiguous span of at least 30 aminoacids of SEQ. ID. NO.:135;

(g) a polypeptide comprising a contiguous span of at least 35 aminoacids of SEQ. ID. NO.:135;

(h) a polypeptide comprising a contiguous span of at least 40 aminoacids of SEQ. ID. NO.:135;

(i) a polypeptide comprising a contiguous span of at least 45 aminoacids of SEQ. ID. NO.:135; and

(j) a polypeptide comprising a contiguous span of at least 50 aminoacids of SEQ. ID. NO.:135; or an allelic variant of said polypeptide;

comprising the step of contacting said known ligand with saidpolypeptide under conditions which allow said binding to occur.

In some embodiments, said known ligand is a modulator of theantilipolytic GPCR. In some embodiments, said known modulator is anagonist of the antilipolytic GPCR. In some embodiments, said knownligand is the modulator of the thirty-sixth aspect. In some embodiments,said known ligand is an antibody specific for the GPCR, or a derivativethereof.

In other preferred embodiments, said method is used to identify whethera candidate compound inhibits said binding of said known ligand to saidpolypeptide, comprising the steps of:

-   -   (a) contacting said polypeptide with said known ligand,        optionally labeled, in the presence or absence of said candidate        compound;    -   (b) detecting the complex between said known ligand and said        polypeptide; and    -   (c) determining whether less of said complex is formed in the        presence of the compound than in the absence of the compound;        wherein said determination is indicative of the candidate        compound being an inhibitor of said binding of said known ligand        to said polypeptide.

In some embodiments, said known ligand is a modulator of theantilipolytic GPCR. In some embodiments, said known modulator is anagonist. In some embodiments, said known ligand is the modulator of thethirty-sixth aspect. In some embodiments, said known ligand is anantibody specific for the GPCR, or a derivative thereof.

In other preferred embodiments, said method is used to identify whethera candidate compound is an inhibitor of said binding of said knownligand to said polypeptide, comprising the steps of:

(a) contacting said polypeptide with said known ligand, optionallylabeled, in the presence separately of a plurality of concentrations ofsaid candidate compound for a time sufficient to allow equilibration ofbinding;

(b) measuring unbound ligand and bound ligand; and

(c) determining K_(i) for the candidate compound;

wherein a K_(i) value of less than 50 uM is indicative of the candidatecompound being an inhibitor of said binding of said known ligand to saidpolypeptide. Preferably said K_(i) value is less than 25 μM, 10 μM, 5μM, 1 μM, 750 μM, 500 nM, 400 nM, 300 μM, 250 μM, 200 nM, 150 nM, 100nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM or 10 nM. Inpreferred embodiments, K_(i) determination is made through nonlinearcurve fitting with the program SCTFIT [De Lean et al. (1982) MolPharmacol 21:5-16; cited in Lorenzen et al. (2001) Mol Pharmacol59:349-357, the disclosures of which are incorporated by referenceherein in their entireties].

In some embodiments, said known ligand is a modulator of theantilipolytic GPCR. In some embodiments, said known modulator is anagonist. In some embodiments, said known ligand is the modulator of thethirty-sixth aspect. In some embodiments, said known ligand is anantibody specific for the GPCR, or a derivative thereof.

In a sixty-eighth aspect, the invention features a method of binding anoptionally labeled affinity reagent specific for an antilipolytic GPCRto said receptor in a biological sample, said receptor comprising theamino acid sequence of SEQ. ID. NO.:135 (hRUP38); or an allelic variant,a biologically active mutant, or a biologically active fragment of saidamino acid sequence, comprising the steps of:

(a) obtaining said biological sample;

(b) contacting the affinity reagent with said receptor in saidbiological sample; and

(c) detecting the complex of said affinity reagent with said receptor.

In some embodiments, the antilipolytic GPCR has the amino acid sequenceof SEQ. ID. NO.:135 (hRUP38); or an allelic variant, a biologicallyactive mutant, or a biologically active fragment of said amino acidsequence.

In some embodiments, the antilipolytic GPCR comprises a biologicallyactive fragment of said amino acid sequence.

In some embodiments, the antilipolytic GPCR is endogenous.

In some embodiments, the antilipolytic GPCR is recombinant.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:135 further substituted at amino acid position 230 withlysine in place of isoleucine.

In preferred embodiments, said G protein is Gi.

In some embodiments, said affinity reagent is a modulator of the GPCR.In some embodiments, said affinity reagent is an agonist of the GPCR. Insome embodiments, said affinity reagent is the modulator of thethirty-sixth aspect. In some embodiments, said affinity reagent is anantibody specific for the GPCR, or a derivative thereof.

In further preferred embodiments, said affinity reagent comprises alabel selected from the group consisting of:

(a) radioisotope;

(b) enzyme; and

(c) fluorophore.

In preferred embodiments, said radioisotope is ³H.

In a sixty-ninth aspect, the invention features the method of thesixty-eighth aspect further comprising the step of comparing the levelof detection of said complex in a first biological sample to a secondlevel of detection of said complex in a second biological sample.

In a seventieth aspect, the invention features the method of thesixty-ninth aspect wherein the relationship between said first andsecond biological samples is selected from the group consisting of:

(a) said second biological sample is a replicate of said firstbiological sample;

(b) said first biological sample was obtained prior to an experimentalintervention

whereas said second biological sample was obtained after theexperimental intervention, from the same individual;

(c) said second biological sample was obtained at a different time pointafter an experimental intervention than was said first biologicalsample, from the same individual;

(d) said second biological sample corresponds to a different subcellularcompartment than does said first biological sample;

(e) said second biological sample represents a different cell type thandoes said first biological sample;

(f) said second biological sample corresponds to a different tissue thandoes said first biological sample;

(g) said second biological sample was obtained from a differentindividual than was said first biological sample;

(h) said second biological sample was obtained at a different point intime than was said first biological sample, from the same individual;

(i) said first biological samples was obtained from a normal individual,whereas said second biological sample was obtained from an individualhaving a metabolic-related disorder;

(j) said first biological sample was obtained from a normal individual,whereas said second biological sample was obtained from an individualhaving a disorder in lipid metabolism;

(k) said first biological sample was obtained before a therapeuticintervention whereas said second biological sample was obtained afterthe therapeutic intervention, from the same individual;

(l) said second biological sample was obtained at a different time pointafter therapeutic intervention than was said first biological sample,from the same individual; and

(m) said first biological sample was not exposed to a compound, whereassaid second biological sample was exposed to said compound.

In a seventy-first aspect, the invention features a method ofidentifying whether a candidate compound is a modulator of anantilipolytic GPCR, said receptor comprising an amino acid sequenceselected from the group consisting of:

(a) SEQ. ID. NO.:24 (hRUP19);

(b) SEQ. ID. NO.:151 (mRUP19); and

(c) SEQ. ID. NO.:157 (rRUP19);

or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence;

comprising the steps of:

(a′) contacting the candidate compound with the receptor;

(b′) determining whether the receptor functionality is modulated;

wherein a change in receptor functionality is indicative of thecandidate compound being a modulator of an antilipolytic GPCR.

In some embodiments, said antilipolytic GPCR is endogenous.

In some preferred embodiments, said antilipolytic GPCR is recombinant.

Preferred said identified modulator binds to said GPCR.

In some embodiments, said contacting is carried out in the presence of aknown ligand of the GPCR. In some embodiments, said known ligand is anagonist of the GPCR.

The invention also relates to a method of identifying whether acandidate compound is a modulator of lipolysis, comprising the steps of:

-   -   (a) contacting the candidate compound with a GPCR comprising an        amino acid sequence selected from the group consisting of:        -   (i) SEQ. ID. NO.:24 (hRUP19);        -   (ii) SEQ. ID. NO.:151 (mRUP19); and        -   (iii) SEQ. ID. NO.:157 (rRUP19);        -   or an allelic variant, a biologically active mutant, or a            biologically active fragment of said amino acid sequence;            and    -   (b) determining whether the receptor functionality is modulated;        wherein a change in receptor functionality is indicative of the        candidate compound being a modulator of lipolysis.

In some embodiments, said GPCR is endogenous.

In some preferred embodiments, said GPCR is recombinant.

Preferred said identified modulator binds to said GPCR.

In some embodiments, said contacting is carried out in the presence of aknown ligand of the GPCR. In some embodiments, said known ligand is anagonist of the GPCR.

The invention also relates to a method of determining whether acandidate compound is a modulator of an antilipolytic GPCR, comprisingthe steps of:

-   -   (a) culturing antilipolytic GPCR-expressing host cells under        conditions that would allow expression of a recombinant        antilipolytic GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant antilipolytic GPCR        comprising an amino acid sequence selected from the group        consisting of:        -   (i) SEQ. ID. NO.:24 (hRUP19);        -   (ii) SEQ. ID. NO.:151 (mRUP19); and        -   (iii) SEQ. ID. NO.:157 (rRUP19);        -   or an allelic variant, a biologically active mutant, or a            biologically active fragment of said amino acid sequence;    -   (b) contacting the antilipolytic GPCR-expressing host cells of        step (a) with the candidate compound;    -   (c) contacting control host cells with the candidate compound of        step (b), wherein said control host cells do not express        recombinant antilipolytic GPCR protein;    -   (d) measuring the modulating effect of the candidate compound        which interacts with the recombinant antilipolytic GPCR from the        host cells of step (a) and control host cells of step (c); and    -   (e) comparing the modulating effect of the test compound on the        host cells and control host cells.

The invention also relates to a method of determining whether acandidate compound is a modulator of an antilipolytic GPCR, comprisingthe steps of:

-   -   (a) culturing antilipolytic GPCR-expressing host cells under        conditions that would allow expression of a recombinant        antilipolytic GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant antilipolytic GPCR        comprising an amino acid sequence selected from the group        consisting of:        -   (i) SEQ. ID. NO.:24 (hRUP19);        -   (ii) SEQ. ID. NO.:151 (mRUP19); and        -   (iii) SEQ. ID. NO.:157 (rRUP19);        -   or an allelic variant, a biologically active mutant, or a            biologically active fragment of said amino acid sequence;    -   (b) contacting a first population of antilipolytic        GPCR-expressing cells of step (a) with a known ligand of said        antilipolytic GPCR;    -   (c) contacting a second population of antilipolytic        GPCR-expressing cells of step (a) with the candidate compound        and with the known antilipolytic GPCR ligand;    -   (d) contacting control host cells with the candidate compound of        step (c), wherein said control host cells do not express        recombinant antilipolytic GPCR protein;    -   (e) measuring the modulating effect of the candidate compound,        which interacts with recombinant antilipolytic GPCR, in the        presence and absence of the known antilipolytic GPCR ligand,        from the cells of step (b), step (c) and step (d); and    -   (f) comparing the modulating effect of the candidate compound as        determined from step (b), step (c) and step (d).

In some embodiments, said ligand is an agonist of the GPCR.

The invention also relates to a method of determining whether acandidate compound is a modulator of an antilipolytic GPCR, comprisingthe steps of:

-   -   (a) culturing antilipolytic GPCR-expressing host cells under        conditions that would allow expression of a recombinant        antilipolytic GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant antilipolytic GPCR        comprising an amino acid sequence selected from the group        consisting of:        -   (i) SEQ. ID. NO.:24 (hRUP19);        -   (ii) SEQ. ID. NO.:151 (mRUP19); and        -   (iii) SEQ. ID. NO.:157 (rRUP19);        -   or an allelic variant, a biologically active mutant, or a            biologically active fragment of said amino acid sequence;    -   (b) contacting a first population of the antilipolytic        GPCR-expressing host cells of step (a) with the candidate        compound;    -   (c) not contacting a second population of the antilipolytic        GPCR-expressing cells of step (a) with the candidate compound of        step (b);    -   (d) contacting control host cells to the candidate compound of        step (b), wherein said control host cells do not express        recombinant antilipolytic GPCR protein;    -   (e) measuring the modulating effect of the candidate compound,        which interacts with recombinant antilipolytic GPCR protein,        from the cells of step (b) and step (c) and from the cells of        step (d); and    -   (f) comparing the modulating effect of the candidate compound as        determined from step (b) and step (c) and from step (d).

In some embodiments, the antilipolytic GPCR has an amino acid sequenceselected from the group consisting of:

(a) SEQ. ID. NO.:24 (hRUP19);

(b) SEQ. ID. NO.:151 (mRUP19); and

(c) SEQ. ID. NO.:157 (rRUP19);

-   -   or an allelic variant, a biologically active mutant, or a        biologically active fragment of said amino acid sequence.

In some embodiments, the antilipolytic GPCR comprises a biologicallyactive fragment of said amino acid sequence.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:24 further substituted at amino acid position 219 withlysine in place of threonine.

In preferred embodiments, said G protein is Gi.

In other preferred embodiments, said determining is through the use of aMelanophore assay.

In other preferred embodiments, said determining is through themeasurement of the level of a second messenger selected from the groupconsisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositoltriphosphate (IP₃), diacylglycerol (DAG), and Ca²⁺. In further preferredembodiments, said second messenger is cAMP. In more preferredembodiments, the level of the cAMP is reduced. In some embodiments, saidmeasurement of cAMP is carried out with membrane comprising said GPCR.

In other preferred embodiments, said determining is through themeasurement of an activity up-regulated or down-regulated by a reductionin intracellular cAMP level. In further preferred embodiments, saiddown-regulated activity is intracellular lipolysis. In other furtherpreferred embodiments, said down-regulated activity is hormone sensitivelipase activity. In other further preferred embodiments, saidup-regulated activity is adiponectin secretion.

In other preferred embodiments, said determining is through CRE-reporterassay. In preferred embodiments, said reporter is luciferase. In someembodiments, said reporter is β-galactosidase.

In other preferred embodiments, said recombinant host cell furthercomprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit andsaid determining is through measurement of intracellular Ca²⁺. Inpreferred embodiments, said Ca²⁺ measurement is carried out by FLIPR.

In other preferred embodiments, said recombinant host cell furthercomprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit andsaid determining is through measurement of intracellular IP₃.

In other preferred embodiments, said determining is through themeasurement of GTPγS binding to membrane comprising said GPCR. Infurther preferred embodiments, said GTPγS is labeled with [³⁵S].

In other preferred embodiments, said method further comprises the stepof comparing the modulation of the receptor caused by the candidatecompound to a second modulation of the receptor caused by contacting thereceptor with a known modulator of the receptor. In some preferredembodiments, said known modulator is an agonist.

In a seventy-second aspect, the invention features a modulator of anantilipolytic GPCR identified according to the method of theseventy-first aspect.

In some preferred embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.More preferably, said modulator is an agonist. In some embodiments, saidmodulator is a partial agonist.

In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 1000 μM in GTP-YS binding assay carried out with membrane fromstably transfected CHO cells expressing recombinant hRUP19 polypeptidehaving the amino acid sequence of SEQ. ID. NO.:24. In some embodiments,said modulator is an agonist with an EC₅₀ of less than 900 μM in saidassay. In some embodiments, said modulator is an agonist with an EC₅₀ ofless than 800 μM in said assay. In some embodiments, said modulator isan agonist with an EC₅₀ of less than 700 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 600μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 550 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 500 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 450 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 400 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 350μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 300 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 250 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 200 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 150 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 100μM in said assay. In some preferred embodiments, said modulator is anagonist with an EC₅₀ in said assay of less than a value selected fromthe interval of 600 μM to 1000 μM.

In some embodiments, said modulator is selective for the GPCR.

In some embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some preferred embodiments, said oral bioavailabilityis at least 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In some preferredembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In highly less preferred embodiments, said modulator is an antibody orderivative thereof.

In a seventy-third aspect, the invention features a method of modulatingthe activity of an antilipolytic GPCR, said receptor comprising an aminoacid sequence selected from the group consisting of:

(a) SEQ. ID. NO.:24 (hRUP19);

(b) SEQ. ID. NO.:151 (mRUP19); and

(c) SEQ. ID. NO.:157 (rRUP19);

or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence;

comprising the step of contacting the receptor with the modulator of theseventy-second aspect.

In some embodiments, the antilipolytic GPCR has an amino acid sequenceselected from the group consisting of:

(a) SEQ. ID. NO.:24 (hRUP19);

(b) SEQ. ID. NO.:151 (mRUP19); and

(c) SEQ. ID. NO.:157 (rRUP19);

or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence.

In some embodiments, the antilipolytic GPCR comprises an active fragmentof said amino acid sequence.

In some embodiments, the antilipolytic GPCR is recombinant.

In some embodiments, the antilipolytic GPCR is endogenous.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:24 further substituted at amino acid position 219 withlysine in place of threonine.

In preferred embodiments, said G protein is Gi.

In some preferred embodiments, said modulator is an agonist.

In preferred embodiments, said modulator is selective for the GPCR.

In other preferred embodiments, said contacting comprises administrationof the modulator to a membrane comprising the receptor.

In other preferred embodiments, said contacting comprises administrationof the modulator to a cell or tissue comprising the receptor.

In other preferred embodiments, said contacting comprises administrationof the modulator to an individual comprising the receptor. In morepreferred embodiments, said individual is a mammal. In other morepreferred embodiments, said mammal is a horse, cow, sheep, pig, cat,dog, rabbit, mouse, rat, non-human primate or human. Yet more preferredis mouse, rat or human. Most preferred is human.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP19polypeptide having the amino acid sequence of SEQ. ID. NO.:24. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said administration is oral.

In preferred embodiments, said modulator is an agonist and saidindividual is in need of prevention of or treatment for ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said modulator is an inverse agonist and saidmetabolic-related disorder relates to a low level of plasma free fattyacids.

In other preferred embodiments, said modulator is an agonist and saidindividual is in need of a change in lipid metabolism selected from thegroup consisting of:

(a) a decrease in the level of plasma triglycerides;

(b) a decrease in the level of plasma free fatty acids;

(c) a decrease in the level of plasma cholesterol;

(d) a decrease in the level of LDL-cholesterol;

(e) an increase in the level of HDL-cholesterol;

(f) a decrease in the total cholesterol/HDL-cholesterol ratio; and

(g) an increase in the level of plasma adiponectin.

In other preferred embodiments, said needed change in lipid metabolismis a decrease in the postprandial increase in plasma free fatty acidsdue to a high fat meal or an inhibition of the progression from impairedglucose tolerance to insulin resistance.

In some embodiments, the modulator is an inverse agonist and the neededchange in lipid metabolism is an increase in the level of plasma freefatty acids.

In other preferred embodiments, said modulator is an agonist and saidindividual is a mouse genetically predisposed to a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure.

In further preferred embodiments, said metabolic-related disorder ishyperlipidemia.

In further preferred embodiments, said method is used to identifywhether said agonist has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering said agonist to the mouse; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering said agonist compared to not administeringsaid agonist; wherein said determination is indicative of said agonisthaving therapeutic efficacy.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other preferred embodiments, said modulator is an agonist and saidindividual is a rat genetically predisposed to a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Infurther preferred embodiments, said metabolic-related disorder ishyperlipidemia.

In further preferred embodiments, said method is used to identifywhether said agonist has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering said agonist to the rat; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering said agonist compared to not administeringsaid agonist; wherein said determination is indicative of said agonisthaving therapeutic efficacy.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In a seventy-fourth aspect, the invention features a method ofpreventing or treating a disorder of lipid metabolism in an individualcomprising contacting a therapeutically effective amount of themodulator of the seventy-second aspect with an antilipolytic GPCR, saidreceptor comprising an amino acid sequence selected from the groupconsisting of:

(a) SEQ. ID. NO.:24 (hRUP19);

(b) SEQ. ID. NO.:151 (mRUP19); and

(c) SEQ. ID. NO.:157 (rRUP19);

or an allelic variant or biologically active fragment of said amino acidsequence.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP19polypeptide having the amino acid sequence of SEQ. ID. NO.:24. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said contacting comprises oraladministration of said modulator to said individual.

In preferred embodiment, said modulator is an agonist and said disorderof lipid metabolism is selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In a seventy-fifth aspect, the invention features a method of preventingor treating a metabolic-related disorder in an individual comprisingcontacting a therapeutically effective amount of the modulator of theseventy-second aspect with an antilipolytic GPCR, said receptorcomprising an amino acid sequence selected from the group consisting of:

(a) SEQ. ID. NO.:24 (hRUP19);

(b) SEQ. ID. NO.:151 (mRUP19); and

(c) SEQ. ID. NO.:157 (rRUP19);

or an allelic variant or biologically active fragment of said amino acidsequence.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP19polypeptide having the amino acid sequence of SEQ. ID. NO.:24. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said contacting comprises oraladministration of said modulator to said individual.

In preferred embodiment, said modulator is an agonist and saidmetabolic-related disorder is selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In a seventy-sixth aspect, the invention features a method of preparinga composition which comprises identifying a modulator of a antilipolyticGPCR and then admixing a carrier and the modulator, wherein themodulator is identifiable by the method of the seventy-first aspect.

In some preferred embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.More preferably, said modulator is an agonist. In some embodiments, saidmodulator is a partial agonist.

In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 1000 μM in GTPγS binding assay carried out with membrane fromstably transfected CHO cells expressing recombinant hRUP19 polypeptidehaving the amino acid sequence of SEQ. ID. NO.:24. In some embodiments,said modulator is an agonist with an EC₅₀ of less than 900 μM in saidassay. In some embodiments, said modulator is an agonist with an EC₅₀ ofless than 800 μM in said assay. In some embodiments, said modulator isan agonist with an EC₅₀ of less than 700 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 600μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 550 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 500 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 450 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 400 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 350μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 300 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 250 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 200 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 150 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 100μM in said assay. In some preferred embodiments, said modulator is anagonist with an EC₅₀ in said assay of less than a value selected fromthe interval of 600 μM to 1000 μM.

In some embodiments, said modulator is selective for the GPCR.

In some embodiments, said modulator is antilipolytic.

In some embodiments, said modulator is orally bioavailable. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to eitherintraperitoneal or intravenous administration. In some preferredembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to either intraperitoneal orintravenous administration.

In an seventy-seventh aspect, the invention features a pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of the modulator of the seventy-secondaspect. In preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP19polypeptide having the amino acid sequence of SEQ. ID. NO.:24. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In a seventy-eighth aspect, the invention features a method of changinglipid metabolism comprising providing or administering to an individualin need of said change said pharmaceutical or physiologically acceptablecomposition of the seventy-seventh aspect, said needed change in lipidmetabolism selected from the group consisting of:

(a) a decrease in the level of plasma triglycerides;

(b) a decrease in the level of plasma free fatty acids;

(c) a decrease in the level of plasma cholesterol;

(d) a decrease in the level of LDL-cholesterol;

(e) an increase in the level of HDL-cholesterol;

(f) a decrease in the total cholesterol/HDL-cholesterol ratio; and

(g) an increase in the level of plasma adiponectin.

In preferred embodiments, a therapeutically effective amount of saidpharmaceutical or physiologically acceptable composition is provided oradministered to said individual.

In some preferred embodiments, said providing or administering of saidpharmaceutical or physiologically acceptable composition is oral.

In other preferred embodiments, said needed change in lipid metabolismis a decrease in the postprandial increase in plasma free fatty acidsdue to a high fat meal or an inhibition of the progression from impairedglucose tolerance to insulin resistance.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In a seventy-ninth aspect, the invention features a method of preventingor treating a metabolic-related disorder comprising providing oradministering to an individual in need of said treatment saidpharmaceutical or physiologically acceptable composition of theseventy-seventh aspect, said metabolic-related disorder selected fromthe group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In preferred embodiments, a therapeutically effective amount of saidpharmaceutical or physiologically acceptable composition is provided oradministered to said individual.

In some preferred embodiments, said providing or administering of saidpharmaceutical or physiologically acceptable composition is oral.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, stroke, Syndrome X, andheart disease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In an eightieth aspect, the invention features a method of using themodulator of the seventy-second aspect for the preparation of amedicament for the treatment of a disorder in lipid metabolism in anindividual.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP19polypeptide having the amino acid sequence of SEQ. ID. NO.:24. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said treatment comprises oraladministration of said medicament to said individual.

In preferred embodiments, said modulator is an agonist and said disorderin lipid metabolism is selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In an eighty-first aspect, the invention features a method of using themodulator of the seventy-second aspect for the preparation of amedicament for the treatment of a metabolic-related disorder in anindividual.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP19polypeptide having the amino acid sequence of SEQ. ID. NO.:24. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said treatment comprises oraladministration of said medicament to said individual.

In preferred embodiments, said modulator is an agonist and saidmetabolic-related disorder is selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In an eighty-second aspect, the invention features a method ofidentifying whether a candidate compound binds to an antilipolytic GPCR,said receptor comprising an amino acid sequence selected from the groupconsisting of:

(a) SEQ. ID. NO.:24 (hRUP19);

(b) SEQ. ID. NO.:151 (mRUP19); and

(c) SEQ. ID. NO.:157 (rRUP19);

or an allelic variant or a biologically active fragment of said aminoacid sequence; comprising the steps of:

(a′) contacting the receptor with a labeled reference compound known tobind to the GPCR in the presence or absence of the candidate compound;and

(b′) determining whether the binding of said labeled reference compoundto the receptor is inhibited in the presence of the candidate compound;wherein said inhibition is indicative of the candidate compound bindingto an antilipolytic GPCR.

In some embodiments, the antilipolytic GPCR comprises a biologicallyactive fragment of said amino acid sequence.

In some embodiments, the antilipolytic GPCR is endogenous.

In some embodiments, the antilipolytic GPCR is recombinant.

In preferred embodiments, said G protein is Gi.

In some preferred embodiments, said reference compound is the modulatorof the seventy-second aspect.

In other embodiments, said reference compound is an antibody specificfor the GPCR, or a derivative thereof.

In preferred embodiments, said reference compound comprises a labelselected from the group consisting of:

(a) radioisotope;

(b) enzyme; and

(c) fluorophore.

In some preferred embodiments, said label is ³H.

In other embodiments, said method further comprises the step ofcomparing the level of inhibition of binding of a labeled firstreference compound by the candidate compound to a second level ofinhibition of binding of said labeled first reference compound by asecond reference compound known to bind to the GPCR.

In an eighty-third aspect, the invention features a method of making amouse genetically predisposed to a metabolic-related disorder selectedfrom the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the step of knocking out the gene encoding the antilipolyticmRUP19 GPCR polypeptide of SEQ. ID. NO.:151.

In some preferred embodiments, said knocking out the gene encoding theantilipolytic mRUP19 GPCR polypeptide of SEQ. ID. NO.:151 is essentiallyrestricted to adipocytes.

In an eighty-fourth aspect, the invention features the knockout mouseaccording to the method of the eighty-third aspect.

In an eighty-fifth aspect, the invention features a method of using theknockout mouse of the eighty-fourth aspect to identify whether acandidate compound has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering the compound to the mouse; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering the compound compared to not administeringthe compound; wherein said determination is indicative of the compoundhaving therapeutic efficacy.

In an eighty-sixth aspect, the invention features a method of making arat genetically predisposed to a metabolic-related disorder selectedfrom the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the step of knocking out the gene encoding the antilipolyticrRUP19 GPCR polypeptide of SEQ. ID. NO.:157.

In some preferred embodiments, said knocking out the gene encoding theantilipolytic rRUP19 GPCR polypeptide of SEQ. ID. NO.:157 is essentiallyrestricted to adipocytes.

In an eighty-seventh aspect, the invention features the knockout rataccording to the method of the eighty-sixth aspect.

In an eighty-eighth aspect, the invention features a method of using theknockout rat of the eighty-seventh aspect to identify whether acandidate compound has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering the compound to the rat; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering the compound compared to not administeringthe compound; wherein said determination is indicative of the compoundhaving therapeutic efficacy.

In an eighty-ninth aspect, the invention features an isolated, purifiedor recombinant RUP19 polynucleotide selected from the group consistingof:

(a) a polynucleotide comprising a contiguous span of at least 75nucleotides of SEQ.ID. NO.:23, SEQ. ID. NO.:150 or SEQ. ID. NO.:156, oran allelic variant of said polynucleotide;

(b) a polynucleotide comprising a contiguous span of at least 150nucleotides of SEQ. ID. NO.:23, SEQ. ID. NO.:150 or SEQ. ID. NO.:156, oran allelic variant of said polynucleotide;

(c) a polynucleotide comprising a contiguous span of at least 250nucleotides of SEQ. ID. NO.:23, SEQ. ID. NO.:150 or SEQ. ID. NO.:156, oran allelic variant of said polynucleotide;

(d) a polynucleotide comprising a contiguous span of at least 350nucleotides of SEQ. ID. NO.:23, SEQ. ID. NO.:150 or SEQ. ID. NO.:156, oran allelic variant of said polynucleotide;

(e) a polynucleotide comprising a contiguous span of at least 500nucleotides of SEQ. ID. NO.:23, SEQ. ID. NO.:150 or SEQ. ID. NO.:156, oran allelic variant of said polynucleotide;

(f) a polynucleotide comprising a contiguous span of at least 750nucleotides of SEQ. ID. NOs.:23, SEQ. ID. NO.:150 or 156, or an allelicvariant of said polynucleotide;

(g) a polynucleotide comprising a contiguous span of at least 1000nucleotides of SEQ. ID. NO.:23, SEQ. ID. NO.:150 or SEQ. ID. NO.:156, oran allelic variant of said polynucleotide;

(h) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 20 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO:151 or SEQ.ID. NO.:157 or an allelic variant of said polypeptide;

(i) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 30 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ.ID. NO.:157 or an allelic variant of said polypeptide;

(j) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 40 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ.ID. NO.:157 or an allelic variant of said polypeptide;

(k) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 50 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ.ID. NO.:157 or an allelic variant of said polypeptide;

(l) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 75 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ.ID. NO.:157 or an allelic variant of said polypeptide;

(m) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 100 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ.ID. NO.:157 or an allelic variant of said polypeptide;

(n) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 150 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ.ID. NO.:157 or an allelic variant of said polypeptide;

(O) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 200 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ.ID. NO.:157 or an allelic variant of said polypeptide;

(p) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 250 amino acids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ.ID. NO.:157 or an allelic variant of said polypeptide; and

(q) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 300 amino acids of SEQ.ID. NO.:24, SEQ. ID. NO.:151 or SEQ.ID. NO.:157 or an allelic variant of said polypeptide.

The invention also relates to an isolated, purified or recombinant RUP19polynucleotide wherein said polynucleotide is selected from the groupconsisting of:

(a) a polynucleotide comprising a nucleotide sequence selected from thegroup consisting of SEQ. ID. NO.:23, SEQ. ID. NO.:150 and SEQ. ID.NO.:156 or an allelic variant of said polynucleotide;

(b) a polynucleotide selected from the group consisting of thepolynucleotide of SEQ. ID. NO.:23, the polynucleotide of SEQ. ID.NO.:150 and the polynucleotide of SEQ. ID. NO.:156, or an allelicvariant of said polynucleotide;

(c) a polynucleotide comprising a nucleotide sequence encoding apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ. ID. NO.:24, SEQ. ID. NO.:151 and SEQ. ID. NO.:157 oran allelic variant of said polypeptide; and

(d) a polynucleotide encoding a polypeptide having an amino acidsequence selected from the group consisting of SEQ. ID. NO.:24, SEQ. ID.NO.:151 and SEQ. ID. NO.:157 or an allelic variant of said polypeptide.

In preferred embodiments, said isolated, purified or recombinantpolynucleotide comprises at least 8 contiguous nucleotides of apolynucleotide of the present invention. In other preferred embodiments,said isolated, purified or recombinant polynucleotide comprises at least10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,500, 600, 700, 800, 900 or 1000 contiguous nucleotides of apolynucleotide of the present invention. Preferably said polynucleotideencodes full-length RUP19 polypeptide or a biologically active fragmentthereof.

The polynucleotides of the present invention include genomicpolynucleotides comprising RUP19 polynucleotides of the invention.

The present invention also relates to a polynucleotide encoding a fusionprotein, wherein said fusion protein comprises an RUP19 polypeptide ofthe invention fused to a heterologous polypeptide. In a preferredembodiment, said polypeptide of the invention is constitutively activeand said heterologous polypeptide is a G protein. In other embodiments,said heterologous polypeptide provides an antigenic epitope. In apreferred embodiment, said heterologous polypeptide provides ahemaglutinin (HA) antigenic epitope. Methods relating to apolynucleotide encoding a fusion protein are well known to those ofordinary skill in the art.

The polynucleotides of the present invention also include variantpolynucleotides at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%identical to an RUP19 polynucleotide of the invention. In a particularlypreferred embodiments, polynucleotide sequence homologies are evaluatedusing the Basic Local Alignment Search Tool (“BLAST”), which is wellknown in the art [See, e.g., Karlin and Altschul, Proc Natl Acad Sci USA(1990) 87:2264-8; Altschul et al., J Mol Biol (1990) 215:403-410;Altschul et al., Nature Genetics (1993) 3:266-72; and Altschul et al.,Nucleic Acids Res (1997) 25:3389-3402; the disclosures of which areincorporated by reference in their entirety].

In further preferred embodiments, the invention features the complementof said polynucleotide.

In a ninetieth aspect, the invention features an isolated, purified orrecombinant RUP19 polypeptide selected from the group consisting of:

(a) a polypeptide comprising a contiguous span of at least 20 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(b) a polypeptide comprising a contiguous span of at least 30 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(c) a polypeptide comprising a contiguous span of at least 40 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(d) a polypeptide comprising a contiguous span of at least 50 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(e) a polypeptide comprising a contiguous span of at least 75 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(f) a polypeptide comprising a contiguous span of at least 100 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(g) a polypeptide comprising a contiguous span of at least 150 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(h) a polypeptide comprising a contiguous span of at least 200 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(i) a polypeptide comprising a contiguous span of at least 250 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157; and

(j) a polypeptide comprising a contiguous span of at least 300 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157; or anallelic variant of said polypeptide.

The invention also relates to an isolated, purified or recombinant RUP19polypeptide wherein said polypeptide is selected from the groupconsisting of:

(a) a polypeptide comprising an amino acid sequence selected from thegroup consisting of SEQ. ID. NO.:24, SEQ. ID. NO.:151 and SEQ. ID.NO.:157; and

(b) a polypeptide selected from the group consisting of the polypeptideof SEQ. ID. NO.:24, the polypeptide of SEQ. ID. NO:151 and thepolypeptide of SEQ. ID. NO.:157; or an allelic variant, a biologicallyactive mutant, or a biologically active fragment of said polypeptide.

In preferred embodiments, said isolated, purified or recombinantpolypeptide comprises at least 6 contiguous amino acids of an RUP19polypeptide of the invention. In further embodiments, said isolated,purified or recombinant polypeptide comprises at least 10, 12, 15, 20,25, 30, 35, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275 or300 contiguous amino acids of a polypeptide of the present invention.Preferably, said polypeptide is full-length RUP19 polypeptide or anactive fragment thereof.

The present invention also relates to a fusion protein, wherein saidfusion protein comprises an RUP19 polypeptide of the invention fused toa heterologous polypeptide. In a preferred embodiment, said polypeptideof the invention is constitutively active and said heterologouspolypeptide is a G protein. In other preferred embodiments, saidheterologous polypeptide provides an antigenic epitope. In particularlypreferred embodiment, said heterologous polypeptide provides ahemaglutinin (HA) antigenic epitope. Methods relating to a fusionprotein are well known to those of ordinary skill in the art.

The polypeptides of the present invention also include variantpolypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to an RUP19 polypeptide of the invention. In aparticularly preferred embodiments, polypeptide sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”), whichis well known in the art [See, e.g., Karlin and Altschul, Proc Natl AcadSci USA (1990) 87:2264-8; Altschul et al., J Mol Biol (1990)215:403-410; Altschul et al., Nature Genetics (1993) 3:266-72; andAltschul et al., Nucleic Acids Res (1997) 25:3389-3402; the disclosuresof which are incorporated by reference in their entirety].

In an ninety-first aspect, the invention features a compositioncomprising, consisting essentially of, or consisting of the RUP19polypeptide of the ninetieth aspect.

In a ninety-second aspect, the invention features a recombinant vector,said vector comprising, consisting essentially of, or consisting of thepolynucleotide of the eighty-ninth aspect. In preferred embodiments,said vector is a targeting vector used in a method of inactivating agene encoding an antilipolytic GPCR of the invention. In other preferredembodiments, said vector is used in a method of transient or stabletransfection.

In particularly preferred embodiment, said vector is an expressionvector for the expression of a antilipolytic GPCR in a recombinant hostcell wherein said expression vector comprises, consists essentially of,or consists of the polynucleotide of the eighty-ninth aspect.

Although a variety of expression vectors are available to those in theart, for purposes of utilization for both the endogenous andnon-endogenous human, mouse and rat GPCRs, it is most preferred that thevector utilized be pCMV. In some alternative embodiments as relates tosaid human, mouse and rat antilipolytic GPCRs, it is preferred that thevector utilized be an adenoviral expression vector.

In a ninety-third aspect, the invention features a prokaryotic oreukaryotic host cell comprising, consisting essentially of, orconsisting of the recombinant vector of the ninety-second aspect. Insome preferred embodiments, said host cell is a eukaryotic embryonicstem cell wherein said vector of the ninety-second aspect has been usedin a method to inactivate a gene encoding an antilipolytic GPCR of theinvention within said cell. In some other preferred embodiments, saidhost cell is a eukaryotic embryonic somatic cell wherein said vector ofthe ninety-second aspect has been used in a method to inactivate a geneencoding an antilipolytic GPCR of the invention within said cell. Inother preferred embodiments, said host cell is prokaryotic and has beentransformed using the vector of the ninety-second aspect. In furtherpreferred embodiments, said host cell is eukaryotic and has beentransiently transfected using the vector of the ninety-second aspect. Inother further preferred embodiments, said host cell is eukaryotic andhas been stably transfected using the vector of the ninety-secondaspect.

In particularly preferred embodiment, said host cell expresses arecombinant antilipolytic GPCR wherein said host cell comprises,consists essentially of, or consists of the expression vector of theninety-second aspect.

A further embodiment includes a prokaryotic or eukaryotic host cellrecombinant for the polynucleotide of the eighty-ninth aspect.

In some embodiments the host cell is eukaryotic, more preferably,mammalian, and more preferably selected from the group consisting of293, 293T, CHO, and COS-7 cells. In other embodiments, the host cell iseukaryotic, more preferably melanophore.

In a ninety-fourth aspect, the invention features a process for theexpression of an antilipolytic GPCR in a recombinant host cellcomprising the steps of:

-   -   (a) transfecting the expression vector of the ninety-second        aspect into a suitable host cell; and    -   (b) culturing the host cells under conditions which allow        expression of the antilipolytic GPCR protein from the expression        vectors.

In a ninety-fifth aspect, the invention features an antibody thatspecifically binds to the polypeptide of the ninetieth aspect. In somepreferred embodiments, the antibody is monoclonal. In some embodiments,the antibody is polyclonal.

In a ninety-sixth aspect, the invention features a method of binding thepolypeptide of the ninetieth aspect to the antibody of the ninety-fifthaspect, comprising contacting said antibody with said polypeptide underconditions in which said antibody can specifically bind to saidpolypeptide.

In a ninety-seventh aspect, the invention features a method of detectingan antilipolytic GPCR polypeptide in a biological sample obtained froman individual comprising the steps of:

(a) obtaining said biological sample from said individual;

(b) contacting said biological sample with the antibody of theninety-fifth aspect; and

(c) detecting the presence or absence of binding of said antibody tosaid biological sample;

wherein a detection of said binding is indicative of the receptorpolypeptide being expressed in said biological sample.

In preferred embodiments, said detecting is through the use of anenzyme-labeled secondary reagent. In other preferred embodiments, saiddetecting is through the use of a fluorophore-labeled secondary reagent.In other preferred embodiments, said detecting is through the use of aradioisotope-labeled secondary reagent. In other embodiments, theantibody is directly labeled with enzyme, fluorophore or radioisotope.

In other preferred embodiments, said biological sample is taken fromadipose, skin or blood.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In further embodiments, said individual has a disorder of lipidmetabolism selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In further embodiments, said individual has a metabolic-related disorderselected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said method further comprises the step ofcomparing the level of detection of said binding for a first individualto the level of detection of said binding for a second individual.

In a ninety-eighth aspect, the invention features a method of detectingexpression of a gene encoding antilipolytic GPCR in a biological sampleobtained from an individual comprising the steps of:

-   -   (a) obtaining said biological sample from said individual;    -   (b) contacting said biological sample with the complementary        polynucleotide of the eighty-ninth aspect, optionally labeled,        under conditions permissive for hybridization; and    -   (c) detecting the presence or absence of said hybridization        between said complementary polynucleotide and an RNA species        within said sample;        wherein a detection of said hybridization is indicative of        expression of said GPCR gene in said biological sample.

Methods of labeling a nucleic acid probe are well known to those ofordinary skill in the art.

In preferred embodiments, the biological sample is taken from adipose,skin or blood.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In preferred embodiments, said individual has a disorder of lipidmetabolism selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In other preferred embodiments, said individual has a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said method further comprises the step ofcomparing the level of detection of said hybridization for a firstindividual to the level of detection of said hybridization for a secondindividual.

In some preferred embodiments, said complementary polynucleotide is aprimer and said hybridization is detected by detecting the presence ofan amplification product comprising the sequence of said primer. In morepreferred embodiments, said method is RT-PCR.

In a ninety-ninth aspect, the invention features a GPCR Fusion Proteinconstruct comprising a constitutively active GPCR and a G protein, saidreceptor comprising an amino acid sequence selected from the groupconsisting of:

(a) SEQ. ID. NO.:24 (hRUP19);

(b) SEQ. ID. NO.:151 (mRUP19); and

(c) SEQ. ID. NO.:157 (rRUP19);

or an allelic variant or a biologically active fragment of said aminoacid sequence.

The invention also relates to a GPCR Fusion Protein construct whereinthe threonine at amino acid position 219 of SEQ. ID. NO.:24 issubstituted by lysine.

In a one hundredth aspect, the invention features a method of binding aknown ligand of RUP19 antilipolytic GPCR to a polypeptide selected fromthe group consisting of:

(a) a polypeptide comprising a contiguous span of at least 6 amino acidsof SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(b) a polypeptide comprising a contiguous span of at least 10 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(c) a polypeptide comprising a contiguous span of at least 15 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(d) a polypeptide comprising a contiguous span of at least 20 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(e) a polypeptide comprising a contiguous span of at least 25 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(f) a polypeptide comprising a contiguous span of at least 30 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(g) a polypeptide comprising a contiguous span of at least 35 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(h) a polypeptide comprising a contiguous span of at least 40 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

(i) a polypeptide comprising a contiguous span of at least 45 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157; and

(j) a polypeptide comprising a contiguous span of at least 50 aminoacids of SEQ. ID. NO.:24, SEQ. ID. NO.:151 or SEQ. ID. NO.:157;

or an allelic variant of said polypeptide;

comprising the step of contacting said known ligand with saidpolypeptide under conditions which allow said binding to occur.

In a some embodiments, said known ligand is a modulator of the GPCR. Insome embodiments, said known ligand is an agonist of the GPCR. In someembodiments, said known ligand is the modulator of the seventy-secondaspect. In some embodiments, said known ligand is an antibody specificfor the GPCR, or a derivative thereof.

In other preferred embodiments, said method is used to identify whethera candidate compound inhibits said binding of said known ligand to saidpolypeptide, comprising the steps of:

-   -   (a) contacting said polypeptide with said known ligand,        optionally labeled, in the presence or absence of said candidate        compound;    -   (b) detecting the complex between said known ligand and said        polypeptide; and    -   (c) determining whether less of said complex is formed in the        presence of the compound than in the absence of the compound;        wherein said determination is indicative of the candidate        compound being an inhibitor of said binding of said known ligand        to said polypeptide.

In a some embodiments, said known ligand is a modulator of the GPCR. Insome embodiments, said known ligand is an agonist of the GPCR. In someembodiments, said known ligand is the modulator of the seventy-secondaspect. In some embodiments, said known ligand is an antibody specificfor the GPCR, or a derivative thereof.

In other preferred embodiments, said method is used to identify whethera candidate compound is an inhibitor of said binding of said knownligand to said polypeptide, comprising the steps of:

(a) contacting said polypeptide with said known ligand, optionallylabeled, in the presence separately of a plurality of concentrations ofsaid candidate compound for a time sufficient to allow equilibration ofbinding;

(b) measuring unbound ligand and bound ligand; and

(c) determining K_(i) for the candidate compound;

wherein a K_(i) value of less than 50 μM is indicative of the candidatecompound being an inhibitor of said binding of said known ligand to saidpolypeptide. Preferably said K_(i) value is less than 25 μM, 10 μM, 5μM, 1 μM, 750 nM, 500 nM, 400 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 mM, 20 nM or 10 nM. Inpreferred embodiments, K_(i) determination is made through nonlinearcurve fitting with the program SCTFIT [De Lean et al. (1982) MolPharmacol 21:5-16; cited in Lorenzen et al. (2001) Mol Pharmacol59:349-357, the disclosures of which are incorporated by referenceherein in their entireties].

In a some embodiments, said known ligand is a modulator of the GPCR. Insome embodiments, said known ligand is an agonist of the GPCR. In someembodiments, said known ligand is the modulator of the seventy-secondaspect. In some embodiments, said known ligand is an antibody specificfor the GPCR, or a derivative thereof.

In a one hundred first aspect, the invention features a method ofbinding an optionally labeled affinity reagent specific for anantilipolytic GPCR to said receptor in a biological sample, saidreceptor comprising an amino acid sequence selected from the groupconsisting of:

(a) SEQ. ID. NO.:24 (hRUP19);

(b) SEQ. ID. NO.:151 (mRUP19); and

(c) SEQ. ID. NO.:157 (rRUP19);

or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence, comprising the steps of:

(a′) obtaining said biological sample;

(b′) contacting the affinity reagent with said receptor in saidbiological sample; and

(c′) detecting the complex of said affinity reagent with said receptor.

In some embodiments, the antilipolytic GPCR has an amino acid sequenceselected from the group consisting of:

(a) SEQ. ID. NO.:24 (hRUP19);

(b) SEQ. ID. NO.:151 (mRUP19); and

(c) SEQ. ID. NO.:157 (rRUP19);

or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence.

In some embodiments, the antilipolytic GPCR comprises an active fragmentof said amino acid sequence.

In some embodiments, the antilipolytic GPCR is endogenous.

In some embodiments, the antilipolytic GPCR is recombinant.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:24 further substituted at amino acid position 219 withlysine in place of threonine.

In preferred embodiments, said G protein is Gi.

In a some embodiments, said affinity reagent is a modulator of the GPCR.In some embodiments, said affinity reagent is an agonist of the GPCR. Insome embodiments, said affinity reagent is the modulator of theseventy-second aspect. In some embodiments, said affinity reagent is anantibody specific for the GPCR, or a derivative thereof.

In further preferred embodiments, said affinity reagent comprises alabel selected from the group consisting of:

(a) radioisotope;

(b) enzyme; and

(c) fluorophore.

In preferred embodiments, said radioisotope is ³H.

In a one hundred second aspect, the invention features the method of theone hundred first aspect further comprising the step of comparing thelevel of detection of said complex in a first biological sample to asecond level of detection of said complex in a second biological sample.

In a one hundred third aspect, the invention features the method of theone hundred second aspect wherein the relationship between said firstand second biological samples is selected from the group consisting of:

(a) said second biological sample is a replicate of said firstbiological sample;

(b) said first biological sample was obtained prior to an experimentalintervention whereas said second biological sample was obtained afterthe experimental intervention, from the same individual;

(c) said second biological sample was obtained at a different time pointafter an experimental intervention than was said first biologicalsample, from the same individual;

(d) said second biological sample corresponds to a different subcellularcompartment than does said first biological sample;

(e) said second biological sample represents a different cell type thandoes said first biological sample;

(f) said second biological sample corresponds to a different tissue thandoes said first biological sample;

(g) said second biological sample was obtained from a differentindividual than was said first biological sample;

(h) said second biological sample was obtained at a different point intime than was said first biological sample, from the same individual;

(i) said first biological samples was obtained from a normal individual,whereas said second biological sample was obtained from an individualhaving a metabolic-related disorder;

(j) said first biological sample was obtained from a normal individual,whereas said second biological sample was obtained from an individualhaving a disorder in lipid metabolism;

(k) said first biological sample was obtained before a therapeuticintervention whereas said second biological sample was obtained afterthe therapeutic intervention, from the same individual;

(l) said second biological sample was obtained at a different time pointafter therapeutic intervention than was said first biological sample,from the same individual; and

(m) said first biological sample was not exposed to a compound, whereassaid second biological sample was exposed to said compound.

In a one hundred fourth aspect, the invention features a method ofidentifying whether a candidate compound is a modulator of anantilipolytic GPCR, said receptor comprising the amino acid sequence ofSEQ. ID. NO.:8 (hRUP11);

or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence;

comprising the steps of:

(a) contacting the candidate compound with the receptor;

(b) determining whether the receptor functionality is modulated;

wherein a change in receptor functionality is indicative of thecandidate compound being a modulator of an antilipolytic GPCR.

In some embodiments, said antilipolytic GPCR is endogenous.

In some preferred embodiments, said antilipolytic GPCR is recombinant.

Preferred said identified modulator binds to said GPCR.

In some embodiments, said contacting is carried out in the presence of aknown ligand of the GPCR. In some embodiments, said known ligand is anagonist of the GPCR.

The invention also relates to a method of identifying whether acandidate compound is a modulator of lipolysis, comprising the steps of:

-   -   (a) contacting the candidate compound with a GPCR comprising the        amino acid sequence of SEQ. ID. NO.:8 (hRUP11); or an allelic        variant, a biologically active mutant, or a biologically active        fragment of said amino acid sequence; and    -   (b) determining whether the receptor functionality is modulated;        wherein a change in receptor functionality is indicative of the        candidate compound being a modulator of lipolysis.

In some embodiments, said GPCR is endogenous.

In some preferred embodiments, said GPCR is recombinant.

Preferred said identified modulator binds to said GPCR.

In some embodiments, said contacting is carried out in the presence of aknown ligand of the GPCR. In some embodiments, said known ligand is anagonist of the GPCR.

The invention also relates to a method of determining whether acandidate compound is a modulator of an antilipolytic GPCR, comprisingthe steps of:

-   -   (a) culturing antilipolytic GPCR-expressing host cells under        conditions that would allow expression of a recombinant        antilipolytic GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant antilipolytic GPCR        comprising the amino acid sequence of SEQ. ID. NO.:8 (hRUP11);        or an allelic variant, a biologically active mutant, or a        biologically active fragment of said amino acid sequence;    -   (b) contacting the antilipolytic GPCR-expressing host cells of        step (a) with the candidate compound;    -   (c) contacting control host cells with the candidate compound of        step (b), wherein said control host cells do not express        recombinant antilipolytic GPCR protein;    -   (d) measuring the modulating effect of the candidate compound        which interacts with the recombinant antilipolytic GPCR from the        host cells of step (a) and control host cells of step (c); and    -   (e) comparing the modulating effect of the test compound on the        host cells and control host cells.

The invention also relates to a method of determining whether acandidate compound is a modulator of an antilipolytic GPCR, comprisingthe steps of:

-   -   (a) culturing antilipolytic GPCR-expressing host cells under        conditions that would allow expression of a recombinant        antilipolytic GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant antilipolytic GPCR        comprising the amino acid sequence of SEQ. ID. NO.:8 (hRUP11);        or an allelic variant, a biologically active mutant, or a        biologically active fragment of said amino acid sequence;    -   (b) contacting a first population of antilipolytic        GPCR-expressing cells of step (a) with a known ligand of said        antilipolytic GPCR;    -   (c) contacting a second population of antilipolytic        GPCR-expressing cells of step (a) with the candidate compound        and with the known antilipolytic GPCR ligand;    -   (d) contacting control host cells with the candidate compound of        step (c), wherein said control host cells do not express        recombinant antilipolytic GPCR protein;    -   (e) measuring the modulating effect of the candidate compound,        which interacts with recombinant antilipolytic GPCR, in the        presence and absence of the known antilipolytic GPCR ligand,        from the cells of step (b), step (c) and step (d); and    -   (f) comparing the modulating effect of the candidate compound as        determined from step (b), step (c) and step (d).

In some embodiments, said known ligand is an agonist of the GPCR.

The invention also relates to a method of determining whether acandidate compound is a modulator of an antilipolytic GPCR, comprisingthe steps of:

-   -   (a) culturing antilipolytic GPCR-expressing host cells under        conditions that would allow expression of a recombinant        antilipolytic GPCR, said host cells being transfected with a        polynucleotide encoding said recombinant antilipolytic GPCR        comprising the amino acid sequence of SEQ. ID. NO.:8 (hRUP11);        or an allelic variant, a biologically active mutant, or a        biologically active fragment of said amino acid sequence;    -   (b) contacting a first population of the antilipolytic        GPCR-expressing host cells of step (a) with the candidate        compound;    -   (c) not contacting a second population of the antilipolytic        GPCR-expressing cells of step (a) with the candidate compound of        step (b);    -   (d) contacting control host cells to the candidate compound of        step (b), wherein said control host cells do not express        recombinant antilipolytic GPCR protein;    -   (e) measuring the modulating effect of the candidate compound,        which interacts with recombinant antilipolytic GPCR protein,        from the cells of step (b) and step (c) and from the cells of        step (d); and    -   (f) comparing the modulating effect of the candidate compound as        determined from step (b) and step (c) and from step (d).

In some embodiments, the antilipolytic GPCR has the amino acid sequenceof SEQ. ID. NO.:8 (hRUP11); or an allelic variant, a biologically activemutant, or a biologically active fragment of said amino acid sequence.

In some embodiments, the antilipolytic GPCR comprises a biologicallyactive fragment of said amino acid sequence.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:8 further substituted at amino acid position 294 withlysine in place of methionine.

In preferred embodiments, said G protein is Gi.

In other preferred embodiments, said determining is through the use of aMelanophore assay.

In other preferred embodiments, said determining is through themeasurement of the level of a second messenger selected from the groupconsisting of cyclic AMP (cAMP), cyclic GMP (cGMP), inositoltriphosphate (IP₃), diacylglycerol (DAG), and Ca²⁺. In further preferredembodiments, said second messenger is cAMP. In more preferredembodiments, the level of the cAMP is reduced. In some embodiments, saidmeasurement of cAMP is carried out with membrane comprising said GPCR.

In other preferred embodiments, said determining is through themeasurement of an activity up-regulated or down-regulated by a reductionin intracellular cAMP level. In further preferred embodiments, saiddown-regulated activity is intracellular lipolysis. In other furtherpreferred embodiments, said down-regulated activity is hormone sensitivelipase activity. In other further preferred embodiments, saidup-regulated activity is adiponectin secretion.

In other preferred embodiments, said determining is through CRE-reporterassay. In preferred embodiments, said reporter is luciferase. In someembodiments, said reporter is β-galactosidase.

In other preferred embodiments, said recombinant host cell furthercomprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit andsaid determining is through measurement of intracellular Ca²⁺. Inpreferred embodiments, said Ca²⁺ measurement is carried out by FLIPR.

In other preferred embodiments, said recombinant host cell furthercomprises promiscuous G alpha 15/16 or chimeric Gq/Gi alpha subunit andsaid determining is through measurement of intracellular IP₃.

In other preferred embodiments, said determining is through themeasurement of GTPγS binding to membrane comprising said GPCR. Infurther preferred embodiments, said GTPγS is labeled with [³⁵S].

In other preferred embodiments, said method further comprises the stepof comparing the modulation of the receptor caused by the candidatecompound to a second modulation of the receptor caused by contacting thereceptor with a known modulator of the receptor. In some preferredembodiments, said known modulator is an agonist.

In a one hundred fifth aspect, the invention features a modulator of anantilipolytic GPCR identified according to the method of the one hundredfourth aspect.

In some preferred embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.More preferably, said modulator is an agonist. In some embodiments, saidmodulator is a partial agonist.

In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 1000 μM in GTPγS binding assay carried out with membrane fromstably transfected CHO cells expressing recombinant hRUP11 polypeptidehaving the amino acid sequence of SEQ. ID. NO.:8. In some embodiments,said modulator is an agonist with an EC₅₀ of less than 900 μM in saidassay. In some embodiments, said modulator is an agonist with an EC₅₀ ofless than 800 μM in said assay. In some embodiments, said modulator isan agonist with an EC₅₀ of less than 700 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 600μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 550 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 500 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 450 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 400 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 350μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 300 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 250 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 200 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 150 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 100μM in said assay. In some preferred embodiments, said modulator is anagonist with an EC₅₀ in said assay of less than a value selected fromthe interval of 600 μM to 1000 μM.

In some embodiments, said modulator is selective for the GPCR.

In some embodiments, said modulator is antilipolytic.

In some embodiments, said modulator is orally bioavailable. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some preferred embodiments, said oral bioavailabilityis at least 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In some preferredembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In highly less preferred embodiments, said modulator is an antibody orderivative thereof.

In a one hundred sixth aspect, the invention features a method ofmodulating the activity of an antilipolytic GPCR, said receptorcomprising the amino acid sequence of SEQ. ID. NO.:8 (hRUP11); or anallelic variant, a biologically active mutant, or a biologically activefragment of said amino acid sequence; comprising the step of contactingthe receptor with the modulator of the one hundred fifth aspect.

In some embodiments, the antilipolytic GPCR has the amino acid sequenceof SEQ. ID. NO.:8 (hRUP11); or an allelic variant, a biologically activemutant, or a biologically active fragment of said amino acid sequence.

In some embodiments, the antilipolytic GPCR comprises an active fragmentof said amino acid sequence.

In some embodiments, the antilipolytic GPCR is endogenous.

In some embodiments, the antilipolytic GPCR is recombinant.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:8 further substituted at amino acid position 294 withlysine in place of methionine.

In preferred embodiments, said G protein is Gi.

In some preferred embodiments, said modulator is an agonist.

In preferred embodiments, said modulator is selective for the GPCR.

In other preferred embodiments, said contacting comprises administrationof the modulator to a membrane comprising the receptor.

In other preferred embodiments, said contacting comprises administrationof the modulator to a cell or tissue comprising the receptor.

In other preferred embodiments, said contacting comprises administrationof the modulator to an individual comprising the receptor. In morepreferred embodiments, said individual is a mammal. In other morepreferred embodiments, said mammal is a horse, cow, sheep, pig, cat,dog, rabbit, mouse, rat, non-human primate or human. Yet more preferredis mouse, rat or human. Most preferred is human.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP11polypeptide having the amino acid sequence of SEQ. ID. NO.:8. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said administration is oral.

In preferred embodiments, said modulator is an agonist and saidindividual is in need of prevention of or treatment for ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said modulator is an inverse agonist and saidmetabolic-related disorder relates to a low level of plasma free fattyacids.

In other preferred embodiments, said modulator is an agonist and saidindividual is in need of a change in lipid metabolism selected from thegroup consisting of:

(a) a decrease in the level of plasma triglycerides;

(b) a decrease in the level of plasma free fatty acids;

(c) a decrease in the level of plasma cholesterol;

(d) a decrease in the level of LDL-cholesterol;

(e) an increase in the level of HDL-cholesterol;

(f) a decrease in the total cholesterol/HDL-cholesterol ratio; and

(g) an increase in the level of plasma adiponectin.

In other preferred embodiments, said needed change in lipid metabolismis a decrease in the postprandial increase in plasma free fatty acidsdue to a high fat meal or an inhibition of the progression from impairedglucose tolerance to insulin resistance.

In some embodiments, the modulator is an inverse agonist and the neededchange in lipid metabolism is an increase in the level of plasma freefatty acids.

In other preferred embodiments, said modulator is an agonist and saidindividual is a mouse genetically predisposed to a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure.

In further preferred embodiments, said metabolic-related disorder ishyperlipidemia.

In further preferred embodiments, said method is used to identifywhether said agonist has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering said agonist to the mouse; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering said agonist compared to not administeringsaid agonist; wherein said determination is indicative of said agonisthaving therapeutic efficacy.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other preferred embodiments, said modulator is an agonist and saidindividual is a rat genetically predisposed to a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Infurther preferred embodiments, said metabolic-related disorder ishyperlipidemia.

In further preferred embodiments, said method is used to identifywhether said agonist has therapeutic efficacy for the treatment of ametabolic-related disorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering said agonist to the rat; and

(b′) determining whether the disorder is prevented, delayed, or madeless severe on administering said agonist compared to not administeringsaid agonist; wherein said determination is indicative of said agonisthaving therapeutic efficacy.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In a one hundred seventh aspect, the invention features a method ofpreventing or treating a disorder of lipid metabolism in an individualcomprising contacting a therapeutically effective amount of themodulator of the one hundred fifth aspect with an antilipolytic GPCR,said receptor comprising the amino acid sequence of SEQ. ID. NO.:8(hRUP11); or an allelic variant or biologically active fragment of saidamino acid sequence.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP11polypeptide having the amino acid sequence of SEQ. ID. NO.:8. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said contacting comprises oraladministration of said modulator to said individual.

In preferred embodiment, said modulator is an agonist and said disorderof lipid metabolism is selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In a one hundred eighth aspect, the invention features a method ofpreventing or treating a metabolic-related disorder in an individualcomprising contacting a therapeutically effective amount of themodulator of the one hundred fifth aspect with an antilipolytic GPCR,said receptor comprising the amino acid sequence of SEQ. ID. NO.:8(hRUP11); or an allelic variant or biologically active fragment of saidamino acid sequence.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTP-YS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP111polypeptide having the amino acid sequence of SEQ. ID. NO.:8. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said contacting comprises oraladministration of said modulator to said individual.

In preferred embodiment, said modulator is an agonist and saidmetabolic-related disorder is selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In a one hundred ninth aspect, the invention features a method ofpreparing a composition which comprises identifying a modulator of anantilipolytic GPCR and then admixing a carrier and the modulator,wherein the modulator is identifiable by the method of the one hundredfourth aspect.

In some preferred embodiments, said modulator is selected from the groupconsisting of agonist, partial agonist, inverse agonist and antagonist.More preferably, said modulator is an agonist. In some embodiments, saidmodulator is a partial agonist.

In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 1000 μM in GTPγS binding assay carried out with membrane fromstably transfected CHO cells expressing recombinant hRUP11 polypeptidehaving the amino acid sequence of SEQ. ID. NO.:8. In some embodiments,said modulator is an agonist with an EC₅₀ of less than 900 μM in saidassay. In some embodiments, said modulator is an agonist with an EC₅₀ ofless than 800 μM in said assay. In some embodiments, said modulator isan agonist with an EC₅₀ of less than 700 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 600μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 550 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 500 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 450 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 400 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 350μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 300 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 250 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 200 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 150 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 100μM in said assay. In some preferred embodiments, said modulator is anagonist with an EC₅₀ in said assay of less than a value selected fromthe interval of 600 μM to 1000 μM.

In some embodiments, said modulator is selective for the GPCR.

In some embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to eitherintraperitoneal or intravenous administration. In some preferredembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to either intraperitoneal orintravenous administration.

In an one hundred tenth aspect, the invention features a pharmaceuticalor physiologically acceptable composition comprising, consistingessentially of, or consisting of the modulator of the one hundred fifthaspect. In preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP11polypeptide having the amino acid sequence of SEQ. ID. NO.:8. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In a one hundred eleventh aspect, the invention features a method ofchanging lipid metabolism comprising providing or administering to anindividual in need of said change said pharmaceutical or physiologicallyacceptable composition of the one hundred tenth aspect, said neededchange in lipid metabolism selected from the group consisting of:

(a) a decrease in the level of plasma triglycerides;

(b) a decrease in the level of plasma free fatty acids;

(c) a decrease in the level of plasma cholesterol;

(d) a decrease in the level of LDL-cholesterol;

(e) an increase in the level of HDL-cholesterol;

(f) a decrease in the total cholesterol/HDL-cholesterol ratio; and

(g) an increase in the level of plasma adiponectin.

In preferred embodiments, a therapeutically effect amount of saidpharmaceutical or physiologically acceptable composition is provided oradministered to said individual.

In some preferred embodiments, said providing or administering of saidpharmaceutical or physiologically acceptable composition is oral.

In other preferred embodiments, said needed change in lipid metabolismis a decrease in the postprandial increase in plasma free fatty acidsdue to a high fat meal or an inhibition of the progression from impairedglucose tolerance to insulin resistance.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In a one hundred twelfth aspect, the invention features a method ofpreventing or treating a metabolic-related disorder comprising providingor administering to an individual in need of said treatment saidpharmaceutical or physiologically acceptable composition of the onehundred tenth aspect, said metabolic-related disorder selected from thegroup consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In preferred embodiments, a therapeutically effect amount of saidpharmaceutical or physiologically acceptable composition is provided oradministered to said individual.

In some preferred embodiments, said providing or administering of saidpharmaceutical or physiologically acceptable composition is oral.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, stroke, Syndrome X, andheart disease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In an one hundred thirteenth aspect, the invention features a method ofusing the modulator of the one hundred fifth aspect for the preparationof a medicament for the treatment of a disorder in lipid metabolism inan individual.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP11polypeptide having the amino acid sequence of SEQ. ID. NO.:8. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said treatment comprises oraladministration of said medicament to said individual.

In preferred embodiments, said modulator is an agonist and said disorderin lipid metabolism is selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In a one hundred fourteenth aspect, the invention features a method ofusing the modulator of the one hundred fifth aspect for the preparationof a medicament for the treatment of a metabolic-related disorder in anindividual.

In some preferred embodiments, said modulator is selective for the GPCR.

In some preferred embodiments, said modulator is orally bioavailable. Insome embodiments, said oral bioavailability is at least 1%, at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% relative to intraperitonealadministration. In some embodiments, said oral bioavailability is atleast 1%, at least 5%, at least 10%, or at least 15% relative tointraperitoneal administration. In some embodiments, said oralbioavailability is at least 1%, at least 5%, at least 10%, at least 15%,at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, orat least 45% relative to intravenous administration. In someembodiments, said oral bioavailability is at least 1%, at least 5%, atleast 10%, or at least 15% relative to intravenous administration.

In some preferred embodiments, said modulator is antilipolytic.

In some preferred embodiments, said modulator is an agonist.

In some preferred embodiments, said modulator is an agonist with an EC₅₀of less than 1000 μM in GTPγS binding assay carried out with membranefrom stably transfected CHO cells expressing recombinant hRUP11polypeptide having the amino acid sequence of SEQ. ID. NO.:8. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 900μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 800 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 700 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 600 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 550 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 500μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 450 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 400 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 350 μM in said assay. In some embodiments, said modulator is anagonist with an EC₅₀ of less than 300 μM in said assay. In someembodiments, said modulator is an agonist with an EC₅₀ of less than 250μM in said assay. In some embodiments, said modulator is an agonist withan EC₅₀ of less than 200 μM in said assay. In some embodiments, saidmodulator is an agonist with an EC₅₀ of less than 150 μM in said assay.In some embodiments, said modulator is an agonist with an EC₅₀ of lessthan 100 μM in said assay. In some preferred embodiments, said modulatoris an agonist with an EC₅₀ in said assay of less than a value selectedfrom the interval of 600 μM to 1000 μM.

In some preferred embodiments, said treatment comprises oraladministration of said medicament to said individual.

In preferred embodiments, said modulator is an agonist and saidmetabolic-related disorder is selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In a one hundred fifteenth aspect, the invention features a method ofidentifying whether a candidate compound binds to an antilipolytic GPCR,said receptor comprising the amino acid sequence of SEQ. ID. NO.:8(hRUP11); or an allelic variant or a biologically active fragment ofsaid amino acid sequence; comprising the steps of:

-   -   (a) contacting the receptor with a labeled reference compound        known to bind to the GPCR in the presence or absence of the        candidate compound; and    -   (b) determining whether the binding of said labeled reference        compound to the receptor is inhibited in the presence of the        candidate compound;        wherein said inhibition is indicative of the candidate compound        binding to an antilipolytic GPCR.

In some embodiments, the antilipolytic acid GPCR comprises abiologically active fragment of said amino acid sequence.

In some embodiments, the antilipolytic GPCR is endogenous.

In some embodiments, the antilipolytic GPCR is recombinant.

In preferred embodiments, said G protein is Gi.

In some preferred embodiments, said reference compound is the modulatorof the one hundred fifth aspect.

In other embodiments, said reference compound is an antibody specificfor the GPCR, or a derivative thereof.

In preferred embodiments, said reference compound comprises a labelselected from the group consisting of:

(a) radioisotope;

(b) enzyme; and

(c) fluorophore.

In some preferred embodiments, said label is ³H.

In other embodiments, said method further comprises the step ofcomparing the level of inhibition of binding of a labeled firstreference compound by the candidate compound to a second level ofinhibition of binding of said labeled first reference compound by asecond reference compound known to bind to the GPCR.

In a one hundred sixteenth aspect, the invention features a method ofmaking a transgenic mouse, comprising the step of engineering said mouseto carry as part of its own genetic material the gene encoding the humanantilipolytic GPCR polypeptide of SEQ. ID. NO.:8 (hRUP11).

In some preferred embodiments, expression of said gene is placed underthe control of an essentially adipocyte specific promoter.

In a one hundred seventeenth aspect, the invention features thetransgenic mouse according to the method of the one hundred sixteenthaspect.

In a one hundred eighteenth aspect, the invention features a method ofusing the transgenic mouse of the one hundred seventeenth aspect toidentify whether an agonist of said human receptor has therapeuticefficacy for the treatment of a disorder of lipid metabolism selectedfrom the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin;

comprising the steps of:

(a′) administering or not administering the agonist to the mouse; and

(b′) determining whether on administering the agonist there is a changeselected from the group consisting of:

-   -   (i) a decrease in the level of plasma triglycerides;    -   (ii) a decrease in the level of plasma free fatty acids;    -   (iii) a decrease in the level of plasma cholesterol;    -   (iv) a decrease in the level of LDL-cholesterol;    -   (v) an increase in the level of HDL-cholesterol;    -   (vi) a decrease in the total cholesterol/HDL-cholesterol ratio;        and    -   (vii) an increase in the level of plasma adiponectin;

wherein said change is indicative of the agonist having therapeuticefficacy.

In a one hundred nineteenth aspect, the invention features a method ofusing the transgenic mouse of the one hundred seventeenth aspect toidentify whether an agonist of said human receptor has therapeuticefficacy for the treatment of a metabolic-related disorder selected fromthe group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering the agonist to the mouse; and

(b′) determining whether on administering the agonist there is a changeselected from the group consisting of:

-   -   (i) a decrease in the level of plasma triglycerides;    -   (ii) a decrease in the level of plasma free fatty acids;    -   (iii) a decrease in the level of plasma cholesterol;    -   (iv) a decrease in the level of LDL-cholesterol;    -   (v) an increase in the level of HDL-cholesterol;    -   (vi) a decrease in the total cholesterol/HDL-cholesterol ratio;        and    -   (vii) an increase in the level of plasma adiponectin;        wherein said change is indicative of the agonist having        therapeutic efficacy.

In a one hundred twentieth aspect, the invention features a method ofmaking a transgenic rat, comprising the step of engineering said rat tocarry as part of its own genetic material the gene encoding the humanantilipolytic GPCR polypeptide of SEQ. ID. NO.:8 (hRUP11).

In some preferred embodiments, expression of said gene is placed underthe control of an essentially adipocyte specific promoter.

In a one hundred twenty-first aspect, the invention features thetransgenic rat according to the method of the one hundred twentiethaspect.

In a one hundred twenty-second aspect, the invention features a methodof using the transgenic rat of the one hundred twenty-first aspect toidentify whether an agonist of said human receptor has therapeuticefficacy for the treatment of a disorder of lipid metabolism selectedfrom the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin;

comprising the steps of:

(a′) administering or not administering the agonist to the rat; and

(b′) determining whether on administering the agonist there is a changeselected from the group consisting of:

-   -   (i) a decrease in the level of plasma triglycerides;    -   (ii) a decrease in the level of plasma free fatty acids;    -   (iii) a decrease in the level of plasma cholesterol;    -   (iv) a decrease in the level of LDL-cholesterol;    -   (v) an increase in the level of HDL-cholesterol;    -   (vi) a decrease in the total cholesterol/HDL-cholesterol ratio;        and    -   (vii) an increase in the level of plasma adiponectin;        wherein said change is indicative of the agonist having        therapeutic efficacy.

In a one hundred twenty-third aspect, the invention features a method ofusing the transgenic rat of the one hundred twenty-first aspect toidentify whether an agonist of said human receptor has therapeuticefficacy for the treatment of a metabolic-related disorder selected fromthe group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes;

comprising the steps of:

(a′) administering or not administering the agonist to the rat; and

(b′) determining whether on administering the agonist there is a changeselected from the group consisting of:

-   -   (i) a decrease in the level of plasma triglycerides;    -   (ii) a decrease in the level of plasma free fatty acids;    -   (iii) a decrease in the level of plasma cholesterol;    -   (iv) a decrease in the level of LDL-cholesterol;    -   (v) an increase in the level of HDL-cholesterol;    -   (vi) a decrease in the total cholesterol/HDL-cholesterol ratio;        and    -   (vii) an increase in the level of plasma adiponectin;        wherein said change is indicative of the agonist having        therapeutic efficacy.

In a one hundred twenty-fourth aspect, the invention features anisolated, purified or recombinant RUP11 polynucleotide selected from thegroup consisting of:

(a) a polynucleotide comprising a contiguous span of at least 75nucleotides of SEQ.ID. NO.:7, or an allelic variant of saidpolynucleotide;

(b) a polynucleotide comprising a contiguous span of at least 150nucleotides of SEQ. ID. NO.:7, or an allelic variant of saidpolynucleotide;

(c) a polynucleotide comprising a contiguous span of at least 250nucleotides of SEQ. ID. NO.:7, or an allelic variant of saidpolynucleotide;

(d) a polynucleotide comprising a contiguous span of at least 350nucleotides of SEQ. ID. NO.:7, or an allelic variant of saidpolynucleotide;

(e) a polynucleotide comprising a contiguous span of at least 500nucleotides of SEQ. ID. NO.:7, or an allelic variant of saidpolynucleotide;

(f) a polynucleotide comprising a contiguous span of at least 750nucleotides of SEQ. ID. NOs.:7, or an allelic variant of saidpolynucleotide;

(g) a polynucleotide comprising a contiguous span of at least 1000nucleotides of SEQ. ID. NO.:7, or an allelic variant of saidpolynucleotide;

(h) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 20 amino acids of SEQ. ID. NO.:8 or an allelic variant ofsaid polypeptide;

(i) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 30 amino acids of SEQ. ID. NO.:8, or an allelic variant ofsaid polypeptide;

(j) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 40 amino acids of SEQ. ID. NO.:8, or an allelic variant ofsaid polypeptide;

(k) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 50 amino acids of SEQ. ID. NO.:8, or an allelic variant ofsaid polypeptide;

(l) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 75 amino acids of SEQ. ID. NO.:8, or an allelic variant ofsaid polypeptide;

(m) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 100 amino acids of SEQ. ID. NO.:8, or an allelic variant ofsaid polypeptide;

(n) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 150 amino acids of SEQ. ID. NO.:8, or an allelic variant ofsaid polypeptide;

(O) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 200 amino acids of SEQ. ID. NO.:8, or an allelic variant ofsaid polypeptide;

(p) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 250 amino acids of SEQ. ID. NO.:8, or an allelic variant ofsaid polypeptide; and

(q) a polynucleotide encoding a polypeptide comprising a contiguous spanof at least 300 amino acids of SEQ. ID. NO.:8, or an allelic variant ofsaid polypeptide.

The invention also relates to an isolated, purified or recombinant RUP11polynucleotide wherein said polynucleotide is selected from the groupconsisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ. ID.NO.:7 or an allelic variant of said nucleotide sequence;

(b) the polynucleotide of SEQ. ID. NO.:7, or an allelic variant of saidpolynucleotide;

(c) a polynucleotide comprising a nucleotide sequence encoding apolypeptide having the amino acid sequence of SEQ. ID. NO.:8 or anallelic variant of said amino acid sequence; and

(d) a polynucleotide encoding a polypeptide having the amino acidsequence of SEQ. ID. NO.:8, or an allelic variant of said polypeptide.

In preferred embodiments, said isolated, purified or recombinantpolynucleotide comprises at least 8 contiguous nucleotides of apolynucleotide of the present invention. In other preferred embodiments,said isolated, purified or recombinant polynucleotide comprises at least10, 12, 15, 18, 20, 25, 28, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,500, 600, 700, 800, 900 or 1000 contiguous nucleotides of apolynucleotide of the present invention. Preferably said polynucleotideencodes full-length RUP11 polypeptide or a biologically active fragmentthereof.

The polynucleotides of the present invention include genomicpolynucleotides comprising RUP11 polynucleotides of the invention.

The present invention also relates to a polynucleotide encoding a fusionprotein, wherein said fusion protein comprises an RUP11 polypeptide ofthe invention fused to a heterologous polypeptide. In a preferredembodiment, said polypeptide of the invention is constitutively activeand said heterologous polypeptide is a G protein. In other embodiments,said heterologous polypeptide provides an antigenic epitope. In apreferred embodiment, said heterologous polypeptide provides ahemaglutinin (HA) antigenic epitope. Methods relating to apolynucleotide encoding a fusion protein are well known to those ofordinary skill in the art.

The polynucleotides of the present invention also include variantpolynucleotides at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99%identical to an RUP11 polynucleotide of the invention. In a particularlypreferred embodiments, polynucleotide sequence homologies are evaluatedusing the Basic Local Alignment Search Tool (“BLAST”), which is wellknown in the art [See, e.g., Karlin and Altschul, Proc Natl Acad Sci USA(1990) 87:2264-8; Altschul et al., J Mol Biol (1990) 215:403-410;Altschul et al., Nature Genetics (1993) 3:266-72; and Altschul et al.,Nucleic Acids Res (1997) 25:3389-3402; the disclosures of which areincorporated by reference in their entirety].

In further preferred embodiments, the invention features the complementof said polynucleotide.

In a one hundred twenty-fifth aspect, the invention features anisolated, purified or recombinant RUP11 polypeptide selected from thegroup consisting of:

(a) a polypeptide comprising a contiguous span of at least 20 aminoacids of SEQ. ID. NO.:8 or an allelic variant of said contiguous span ofamino acids;

(b) a polypeptide comprising a contiguous span of at least 30 aminoacids of SEQ. ID. NO.:8 or an allelic variant of said contiguous span ofamino acids;

(c) a polypeptide comprising a contiguous span of at least 40 aminoacids of SEQ. ID. NO.:8 or an allelic variant of said contiguous span ofamino acids;

(d) a polypeptide comprising a contiguous span of at least 50 aminoacids of SEQ. ID. NO.:8 or an allelic variant of said contiguous span ofamino acids;

(e) a polypeptide comprising a contiguous span of at least 75 aminoacids of SEQ. ID. NO.:8 or an allelic variant of said contiguous span ofamino acids;

(f) a polypeptide comprising a contiguous span of at least 100 aminoacids of SEQ. ID. NO.:8 or an allelic variant of said contiguous span ofamino acids;

(g) a polypeptide comprising a contiguous span of at least 150 aminoacids of SEQ. ID. NO.:8 or an allelic variant of said contiguous span ofamino acids;

(h) a polypeptide comprising a contiguous span of at least 200 aminoacids of SEQ. ID. NO.:8 or an allelic variant of said contiguous span ofamino acids;

(i) a polypeptide comprising a contiguous span of at least 250 aminoacids of SEQ. ID. NO.:8 or an allelic variant of said contiguous span ofamino acids; and

(j) a polypeptide comprising a contiguous span of at least 300 aminoacids of SEQ. ID. NO.:8 or an allelic variant of said contiguous span ofamino acids.

The invention also relates to an isolated, purified or recombinant RUP11polypeptide wherein said polypeptide is selected from the groupconsisting of:

(a) a polypeptide comprising the amino acid sequence of SEQ. ID. NO.:8or an allelic variant or a biologically active mutant of said amino acidsequence; and

(b) the polypeptide having the amino acid sequence of SEQ. ID. NO.:8 oran allelic variant or a biologically active mutant of said amino acidsequence; or a biologically active fragment of said polypeptide.

In preferred embodiments, said isolated, purified or recombinantpolypeptide comprises at least 6 contiguous amino acids of an RUP11polypeptide of the invention. In further embodiments, said isolated,purified or recombinant polypeptide comprises at least 10, 12, 15, 20,25, 30, 35, 40, 50, 60, 75, 100, 125, 150, 175, 200, 225, 250, 275 or300 contiguous amino acids of a polypeptide of the present invention.Preferably, said polypeptide is full-length RUP11 polypeptide or anactive fragment thereof.

The present invention also relates to a fusion protein, wherein saidfusion protein comprises an RUP11 polypeptide of the invention fused toa heterologous polypeptide. In a preferred embodiment, said polypeptideof the invention is constitutively active and said heterologouspolypeptide is a G protein. In other preferred embodiments, saidheterologous polypeptide provides an antigenic epitope. In particularlypreferred embodiment, said heterologous polypeptide provides ahemaglutinin (HA) antigenic epitope. Methods relating to a fusionprotein are well known to those of ordinary skill in the art.

The polypeptides of the present invention also include variantpolypeptides at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to an RUP11 polypeptide of the invention. In aparticularly preferred embodiments, polypeptide sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”), whichis well known in the art [See, e.g., Karlin and Altschul, Proc Natl AcadSci USA (1990) 87:2264-8; Altschul et al., J Mol Biol (1990)215:403-410; Altschul et al., Nature Genetics (1993) 3:266-72; andAltschul et al., Nucleic Acids Res (1997) 25:3389-3402; the disclosuresof which are incorporated by reference in their entirety].

In an one hundred twenty-sixth aspect, the invention features acomposition comprising, consisting essentially of, or consisting of theRUP11 polypeptide of the one hundred twenty-fifth aspect.

In a one hundred twenty-seventh aspect, the invention features arecombinant vector, said vector comprising, consisting essentially of,or consisting of the polynucleotide of the one hundred twenty-fourthaspect. In some embodiments, said vector is a targeting vector used in amethod of inactivating a gene encoding an antilipolytic GPCR of theinvention. In some preferred embodiments, said vector is used in amethod of transient or stable transfection. In other preferredembodiments, said vector is used in a method of transgenic expression.

In particularly preferred embodiment, said vector is an expressionvector for the expression of a an antilipolytic GPCR in a recombinanthost cell wherein said expression vector comprises, consists essentiallyof, or consists of the polynucleotide of the one hundred twenty-fourthaspect.

Although a variety of expression vectors are available to those in theart, for purposes of utilization for both the endogenous andnon-endogenous human, mouse and rat GPCRs, it is most preferred that thevector utilized be pCMV. In some alternative embodiments as relates tosaid human, mouse and rat antilipolytic GPCRs, it is preferred that thevector utilized be an adenoviral expression vector.

In a one hundred twenty-eighth aspect, the invention features aprokaryotic or eukaryotic host cell comprising, consisting essentiallyof, or consisting of the recombinant vector of the one hundredtwenty-seventh aspect. In some embodiments, said host cell is aeukaryotic embryonic stem cell wherein said vector of the one hundredtwenty-seventh aspect has been used in a method to inactivate a geneencoding an antilipolytic GPCR of the invention within said cell. Insome embodiments, said host cell is a eukaryotic embryonic somatic cellwherein said vector of the one hundred twenty-seventh aspect has beenused in a method to inactivate a gene encoding an antilipolytic GPCR ofthe invention within said cell. In some preferred embodiments, said hostcell is derived from a mouse or rat made transgenic for a human RUP11antilipolytic GPCR of the invention. In other preferred embodiments,said host cell is prokaryotic and has been transformed using the vectorof the one hundred twenty-seventh aspect. In further preferredembodiments, said host cell is eukaryotic and has been transientlytransfected using the vector of the one hundred twenty-seventh aspect.In other further preferred embodiments, said host cell is eukaryotic andhas been stably transfected using the vector of the one hundredtwenty-seventh aspect.

In particularly preferred embodiment, said host cell expresses arecombinant antilipolytic GPCR wherein said host cell comprises,consists essentially of, or consists of the expression vector of the onehundred twenty-seventh aspect.

A further embodiment includes a prokaryotic or eukaryotic host cellrecombinant for the polynucleotide of the one hundred twenty-fourthaspect.

In some embodiments the host cell is eukaryotic, more preferably,mammalian, and more preferably selected from the group consisting of293, 293T, CHO, and COS-7 cells. In other embodiments, the host cell iseukaryotic, more preferably melanophore.

In a one hundred twenty-ninth aspect, the invention features a processfor the expression of a antilipolytic GPCR in a recombinant host cellcomprising the steps of:

(a) transfecting the expression vector of the one hundred twenty-seventhaspect into a suitable host cell; and

(b) culturing the host cells under conditions which allow expression ofthe antilipolytic GPCR protein from the expression vectors.

In a one hundred thirtieth aspect, the invention features an antibodythat specifically binds to the polypeptide of the one hundredtwenty-fifth aspect. In some preferred embodiments, the antibody ismonoclonal. In some embodiments, the antibody is polyclonal.

In a one hundred thirty-first aspect, the invention features a method ofbinding the polypeptide of the one hundred twenty-fifth aspect to theantibody of the one hundred thirtieth aspect, comprising contacting saidantibody with said polypeptide under conditions in which said antibodycan specifically bind to said polypeptide.

In a one hundred thirty-second aspect, the invention features a methodof detecting an antilipolytic GPCR polypeptide in a biological sampleobtained from an individual comprising the steps of:

-   -   (a) obtaining said biological sample from said individual;    -   (b) contacting said biological sample with the antibody of the        one hundred thirtieth aspect; and    -   (c) detecting the presence or absence of binding of said        antibody to said biological sample;        wherein a detection of said binding is indicative of the        receptor polypeptide being expressed in said biological sample.

In preferred embodiments, said detecting is through the use of anenzyme-labeled secondary reagent. In other preferred embodiments, saiddetecting is through the use of a fluorophore-labeled secondary reagent.In other preferred embodiments, said detecting is through the use of aradioisotope-labeled secondary reagent. In other embodiments, theantibody is directly labeled with enzyme, fluorophore or radioisotope.

In other preferred embodiments, said biological sample is taken fromadipose, skin or blood.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In further embodiments, said individual has a disorder of lipidmetabolism selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In further embodiments, said individual has a metabolic-related disorderselected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said method further comprises the step ofcomparing the level of detection of said binding for a first individualto the level of detection of said binding for a second individual.

In a one hundred thirty-third aspect, the invention features a method ofdetecting expression of a gene encoding an antilipolytic GPCR in abiological sample obtained from an individual comprising the steps of:

-   -   (a) obtaining said biological sample from said individual;    -   (b) contacting said biological sample with the complementary        polynucleotide of the one hundred twenty-fourth aspect,        optionally labeled, under conditions permissive for        hybridization; and    -   (c) detecting the presence or absence of said hybridization        between said complementary polynucleotide and an RNA species        within said sample;

wherein a detection of said hybridization is indicative of expression ofsaid GPCR gene in said biological sample.

In preferred embodiments, the biological sample is taken from adipose,skin or blood.

In preferred embodiments, said individual is a mammal. In more preferredembodiments, said mammal is a horse, cow, sheep, pig, cat, dog, rabbit,mouse, rat, non-human primate or human. Yet more preferred is mouse, rator human. Most preferred is human.

In preferred embodiments, said individual has a disorder of lipidmetabolism selected from the group consisting of:

(a) elevated level of plasma triglycerides;

(b) elevated level of plasma free fatty acids;

(c) elevated level of plasma cholesterol;

(d) elevated level of LDL-cholesterol;

(e) reduced level of HDL-cholesterol;

(f) elevated total cholesterol/HDL-cholesterol ratio; and

(g) reduced level of plasma adiponectin.

In other preferred embodiments, said disorder in lipid metabolism is anelevated postprandial increase in plasma free fatty acids due to a highfat meal or a progression from impaired glucose tolerance to insulinresistance.

In other preferred embodiments, said individual has a metabolic-relateddisorder selected from the group consisting of:

(a) dyslipidemia;

(b) atherosclerosis;

(c) coronary heart disease;

(d) stroke;

(e) insulin resistance; and

(f) type 2 diabetes.

In other preferred embodiments, said metabolic-related disorder isselected from the group consisting of obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. In otherpreferred embodiments, said metabolic-related disorder ishyperlipidemia.

In other embodiments, said method further comprises the step ofcomparing the level of detection of said hybridization for a firstindividual to the level of detection of said hybridization for a secondindividual.

In some preferred embodiments, said complementary polynucleotide is aprimer and said hybridization is detected by detecting the presence ofan amplification product comprising the sequence of said primer. In morepreferred embodiments, said method is RT-PCR.

In a one hundred thirty-fourth aspect, the invention features a GPCRFusion Protein construct comprising a constitutively active GPCR and a Gprotein, said receptor comprising the amino acid sequence of SEQ. ID.NO.:8 (hRUP11) or an allelic variant or a biologically active fragmentof said amino acid sequence.

The invention also relates to a GPCR Fusion Protein construct whereinthe methionine at amino acid position 294 of SEQ. ID. NO.:8 issubstituted by lysine.

In a one hundred thirty-fifth aspect, the invention features a method ofbinding a known ligand of RUP11 antilipolytic GPCR to a polypeptideselected from the group consisting of:

(a) a polypeptide comprising a contiguous span of at least 6 amino acidsof SEQ. ID. NO.:8;

(b) a polypeptide comprising a contiguous span of at least 10 aminoacids of SEQ. ID. NO.:8;

(c) a polypeptide comprising a contiguous span of at least 15 aminoacids of SEQ. ID. NO.:8;

(d) a polypeptide comprising a contiguous span of at least 20 aminoacids of SEQ. ID. NO.:8;

(e) a polypeptide comprising a contiguous span of at least 25 aminoacids of SEQ. ID. NO.:8;

(f) a polypeptide comprising a contiguous span of at least 30 aminoacids of SEQ. ID. NO.:8;

(g) a polypeptide comprising a contiguous span of at least 35 aminoacids of SEQ. ID. NO.:8;

(h) a polypeptide comprising a contiguous span of at least 40 aminoacids of SEQ. ID. NO.:8;

(i) a polypeptide comprising a contiguous span of at least 45 aminoacids of SEQ. ID. NO.:8; and

(j) a polypeptide comprising a contiguous span of at least 50 aminoacids of SEQ. ID. NO.:8; or an allelic variant of said polypeptide;

comprising the step of contacting said known ligand with saidpolypeptide under conditions which allow said binding to occur.

In some embodiments, said known ligand is a modulator of the GPCR. Insome embodiments, said known ligand is an agonist of the GPCR. In someembodiments, said known ligand is the modulator of the one hundred fifthaspect. In some embodiments, said known ligand is an antibody specificfor the GPCR, or a derivative thereof.

In other preferred embodiments, said method is used to identify whethera candidate compound inhibits said binding of said known ligand to saidpolypeptide, comprising the steps of:

-   -   (a) contacting said polypeptide with said known ligand,        optionally labeled, in the presence or absence of said candidate        compound;    -   (b) detecting the complex between said known ligand and said        polypeptide; and    -   (c) determining whether less of said complex is formed in the        presence of the    -   compound than in the absence of the compound; wherein said        determination is indicative of the candidate compound being an        inhibitor of said binding of said known ligand to said        polypeptide.

In some embodiments, said known ligand is a modulator of the GPCR. Insome embodiments, said known ligand is an agonist of the GPCR. In someembodiments, said known ligand is the modulator of the one hundred fifthaspect. In some embodiments, said known ligand is an antibody specificfor the GPCR, or a derivative thereof.

In other preferred embodiments, said method is used to identify whethera candidate compound is an inhibitor of said binding of said knownligand to said polypeptide, comprising the steps of:

(a) contacting said polypeptide with said known ligand, optionallylabeled, in the presence separately of a plurality of concentrations ofsaid candidate compound for a time sufficient to allow equilibration ofbinding;

(b) measuring unbound ligand and bound ligand; and

(c) determining K_(i) for the candidate compound;

wherein a K_(i) value of less than 50 uM is indicative of the candidatecompound being an inhibitor of said binding of said known ligand to saidpolypeptide. Preferably said K_(i) value is less than 25 μM, 10 μM, 5μM, 1 μM, 750 nM, 500 nM, 400 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100nM, 90 nM, 80 nM, 70 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM or 10 nM. Inpreferred embodiments, K_(i) determination is made through nonlinearcurve fitting with the program SCTFIT [De Lean et al. (1982) MolPharmacol 21:5-16; cited in Lorenzen et al. (2001) Mol Pharmacol59:349-357, the disclosures of which are incorporated by referenceherein in their entireties].

In some embodiments, said known ligand is a modulator of the GPCR. Insome embodiments, said known ligand is an agonist of the GPCR. In someembodiments, said known ligand is the modulator of the one hundred fifthaspect. In some embodiments, said known ligand is an antibody specificfor the GPCR, or a derivative thereof.

In a one hundred thirty-six aspect, the invention features a method ofbinding an optionally labeled affinity reagent specific for anantilipolytic GPCR to said receptor in a biological sample, saidreceptor comprising the amino acid sequence of SEQ. ID. NO.:8 (hRUP11);or an allelic variant, a biologically active mutant, or a biologicallyactive fragment of said amino acid sequence, comprising the steps of:

(a) obtaining said biological sample;

(b) contacting the affinity reagent with said receptor in saidbiological sample; and

(c) detecting the complex of said affinity reagent with said receptor.

In some embodiments, the antilipolytic GPCR has the amino acid sequenceof SEQ. ID. NO.:8 (hRUP11); or an allelic variant, a biologically activemutant, or a biologically active fragment of said amino acid sequence.

In some embodiments, the antilipolytic GPCR comprises a biologicallyactive fragment of said amino acid sequence.

In some embodiments, the antilipolytic GPCR is endogenous.

In some embodiments, the antilipolytic GPCR is recombinant.

In some embodiments, said biologically active mutant is CART or EFA. Inpreferred embodiments, said CART mutant has the amino acid sequence ofSEQ. ID. NO.:8 further substituted at amino acid position 294 withlysine in place of methionine.

In preferred embodiments, said G protein is Gi.

In some embodiments, said affinity reagent is a modulator of the GPCR.In some embodiments, said affinity reagent is an agonist of the GPCR. Insome embodiments, said affinity reagent is the modulator of the onehundred fifth aspect. In some embodiments, said affinity reagent is anantibody specific for the GPCR, or a derivative thereof.

In further preferred embodiments, said affinity reagent comprises alabel selected from the group consisting of:

(a) radioisotope;

(b) enzyme; and

(c) fluorophore.

In preferred embodiments, said radioisotope is ³H.

In a one hundred thirty-seventh aspect, the invention features themethod of the one hundred thirty-sixth aspect further comprising thestep of comparing the level of detection of said complex in a firstbiological sample to a second level of detection of said complex in asecond biological sample.

In a one hundred thirty-eighth aspect, the invention features the methodof the one hundred thirty-seventh aspect wherein the relationshipbetween said first and second biological samples is selected from thegroup consisting of:

(a) said second biological sample is a replicate of said firstbiological sample;

(b) said first biological sample was obtained prior to an experimentalintervention whereas said second biological sample was obtained afterthe experimental intervention, from the same individual;

(c) said second biological sample was obtained at a different time pointafter an experimental intervention than was said first biologicalsample, from the same individual;

(d) said second biological sample corresponds to a different subcellularcompartment than does said first biological sample;

(e) said second biological sample represents a different cell type thandoes said first biological sample;

(f) said second biological sample corresponds to a different tissue thandoes said first biological sample;

(g) said second biological sample was obtained from a differentindividual than was said first biological sample;

(h) said second biological sample was obtained at a different point intime than was said first biological sample, from the same individual;

(i) said first biological samples was obtained from a normal individual,whereas said second biological sample was obtained from an individualhaving a metabolic-related disorder;

(j) said first biological sample was obtained from a normal individual,whereas said second biological sample was obtained from an individualhaving a disorder in lipid metabolism;

(k) said first biological sample was obtained before a therapeuticintervention whereas said second biological sample was obtained afterthe therapeutic intervention, from the same individual;

(l) said second biological sample was obtained at a different time pointafter therapeutic intervention than was said first biological sample,from the same individual; and

(m) said first biological sample was not exposed to a compound, whereassaid second biological sample was exposed to said compound.

In a one hundred thirty-ninth aspect, the invention features an isolatedEFA-hRUP25 polynucleotide selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ. ID.NO.:158;

(b) a polynucleotide having the nucleotide sequence of SEQ. ID. NO.:158;

(c) a polynucleotide comprising a polynucleotide encoding thepolypeptide having the amino acid sequence of SEQ. ID. NO.:159 or abiologically active fragment of said polypeptide; and

(d) a polynucleotide encoding the polypeptide having the amino acidsequence of SEQ. ID. NO.:159 or a biologically active fragment of saidpolypeptide.

In a one hundred fortieth aspect, the invention features an isolatedEFA-hRUP25 polypeptide selected from the group consisting of:

(a) a polypeptide comprising the amino acid sequence of SEQ. ID.NO.:159, or a biologically active fragment of said polypeptide; and

(b) a polypeptide having the amino acid sequence of SEQ. ID. NO.:159, ora biologically active fragment of said polypeptide.

In an one hundred forty-first aspect, the invention features acomposition comprising, consisting essentially of, or consisting of theEFA-hRUP25 polypeptide of the one hundred fortieth aspect.

In a one hundred forty-second aspect, the invention features arecombinant vector comprising the polynucleotide of the one hundredthirty-ninth aspect. In some preferred embodiments, said vector is usedin a method of transient or stable transfection.

In particularly preferred embodiment, said vector is an expressionvector for the expression of an EFA-hRUP25 nicotinic acid GPCR in arecombinant host cell wherein said expression vector comprises, consistsessentially of, or consists of the polynucleotide of the one hundredthirty-ninth aspect.

Although a variety of expression vectors are available to those in theart, it is most preferred that the vector utilized be pCMV. In somealternative embodiments as relates to EFA-hRUP25 nicotinic acid GPCR, itis preferred that the vector utilized be an adenoviral expressionvector.

In a one hundred forty-third aspect, the invention features aprokaryotic or eukaryotic host cell comprising, consisting essentiallyof, or consisting of the recombinant vector of the one hundredforty-second aspect. In some embodiments, said host cell is prokaryoticand has been transformed using the vector of the one hundredforty-second aspect. In some embodiments, said host cell is eukaryoticand has been transiently transfected using the vector of the one hundredforty-second aspect. In some preferred embodiments, said host cell iseukaryotic and has been stably transfected using the vector of the onehundred forty-second aspect.

In particularly preferred embodiment, said host cell expresses arecombinant EFA-hRUP25 nicotinic acid GPCR wherein said host cellcomprises, consists essentially of, or consists of the expression vectorof the one hundred forty-second aspect.

A further embodiment includes a prokaryotic or eukaryotic host cellrecombinant for the polynucleotide of the one hundred thirty-ninthaspect.

In some embodiments the host cell is eukaryotic, more preferably,mammalian, and more preferably selected from the group consisting of293, 293T, CHO, and COS-7 cells. In other embodiments, the Host Cell iseukaryotic, more preferably melanophore.

In a one hundred forty-fourth aspect, the invention features a processfor the expression of an EFA-hRUP25 nicotinic acid GPCR in a recombinanthost cell comprising the steps of:

-   -   (a) transfecting the expression vector of the one hundred        forty-second aspect into a suitable host cell; and    -   (b) culturing the host cells under conditions which allow        expression of the EFA-hRUP25 nicotinic acid GPCR protein from        the expression vectors.

In a one hundred forty-fifth aspect, the invention features a method ofmaking an EFA mutant of an endogenous GPCR polypeptide havingconstitutive activity, comprising the steps of:

(a) introducing 1, 2, 3, 4, or 5 substitutions, insertions, or deletionsinto the amino acid sequence of the endogenous GPCR polypeptide;

(b) measuring the activity of the mutant GPCR of (a) in the absence ofagonist and in the presence of a known agonist;

(c) measuring the activity of the endogenous GPCR in the absence ofagonist and in the presence of said known agonist; and

(d) comparing (b) and (c);

wherein a determination that the agonist screening window of (b) is atleast 20% greater than that of (c) identifies the mutant resulting from(a) to be an EFA mutant of the endogenous GPCR.

In some embodiments, said number of substitutions, insertions, ordeletions is 1.

In some embodiments, said number of substitutions, insertions, ordeletions is 1 or 2.

In some embodiments, said number of substitutions, insertions, ordeletions is 1, 2 or 3.

In some embodiments, said number of substitutions, insertions, ordeletions is 1, 2, 3 or 4.

In more preferred embodiments, said number of substitutions, insertions,or deletions is 1, 2, 3, 4 or 5.

Applicant reserves the right to exclude any one or more candidatecompounds from any of the embodiments of the invention. Applicant alsoreserves the right to exclude any one or more modulators from any of theembodiments of the invention, including but not limited to nicotinicacid or any analog or derivative thereof. Applicant further reserves theright to exclude any polynucleotide or polypeptide from any of theembodiments of the invention. Applicant additionally reserves the rightto exclude any metabolic-related disorder or any disorder of lipidmetabolism from any of the embodiments of the invention.

Throughout this application, various publications, patents and publishedpatent applications are cited. The disclosures of these publications,patents and published patent applications referenced in this applicationare hereby incorporated by reference in their entirety into the presentdisclosure. Citation herein by Applicant of a publication, patent, orpublished patent application is not an admission by Applicant of saidpublication, patent, or published patent application as prior art.

Modifications and extension of the disclosed inventions that are withinthe purview of the skilled artisan are encompassed within the abovedisclosure and the claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. FIG. 1 depicts second messenger IP₃ production from endogenousversion hRUP12 (“HRUP12”) as compared with the control (“CMV”).

FIG. 2. FIG. 2 depicts the results of a second messenger cell-basedcyclic AMP assay providing comparative results for constitutivesignaling of endogenous hRUP13 (“hRUP13”) and a control vector (“CMV”).

FIG. 3. FIG. 3 depicts the signal measured comparing CMV, endogenoushRUP13 (“hRUP13 wt”) and non-endogenous, constitutively activated hRUP13(“hRUP13(A268K)”), utilizing 8XCRE-Luc reporter plasmid.

FIG. 4. FIG. 4 depicts the results of a [³⁵S]GTPγS assay providingcomparative results for constitutive signaling by hRUP13:Gs FusionProtein (“hRUP13-Gs”) and a control vector (“CMV”).

FIG. 5. FIG. 5 depicts the signal measured comparing CMV, endogenoushRUP14 (“hRUP14 wt”) and non-endogenous, constitutively activated hRUP13(“hRUP14(L246K)”), utilizing 8XCRE-Luc reporter plasmid.

FIG. 6. FIG. 6 depicts the signal measured comparing CMV, endogenoushRUP15 (“hRUP15 wt”) and non-endogenous, constitutively activated hRUP15(“hRUP 15(A398K)”), utilizing 8XCRE-Luc reporter plasmid.

FIG. 7. FIG. 7 depicts the results of a second messenger cell-basedcyclic AMP assay providing comparative results for constitutivesignaling of endogenous hRUP15 (“hRUP15 wt”), non-endogenous,constitutively activated version of hRUP15 (“hRUP15(A398K)”) and acontrol vector (“CMV”).

FIG. 8. FIG. 8 depicts the results of a [³⁵S]GTPγS assay providingcomparative results for constitutive signaling by hRUP15:Gs FusionProtein (“hRUP15-Gs”) and a control vector (“CMV”).

FIG. 9. FIG. 9 depicts second messenger IP₃ production from endogenousversion hRUP17 (“hRUP17”) as compared with the control (“CMV”).

FIG. 10. FIG. 10 depicts messenger IP₃ production from endogenousversion hRUP21 (“hRUP21”) as compared with the control (“CMV”).

FIG. 11. FIG. 11 depicts the signal measured comparing CMV, endogenoushRUP23 (“hRUP23 wt”) and non-endogenous, constitutively activated hRUP23(“hRUP23(W275K)”), utilizing 8XCRE-Luc reporter plasmid.

FIG. 12. FIG. 12 depicts results from a primary screen of severalcandidate compounds against hRUP13; results for “Compound A” areprovided in well A2 and “Compound “B” are provided in well G9.

FIGS. 13A-C. FIGS. 13A and 13B are histograms representing relativeexpression levels of hRUP25 (FIG. 13A) and hRUP38 (FIG. 13B) detected indifferent human tissues via DNA microarray. The horizontal axis displaysthe different tissues, identified in vertical text above the bar. Thevertical axis indicates level of expression of either hRUP25 (FIG. 13A)or hRUP38 (FIG. 13B). In FIG. 13A and FIG. 13B, note the high level ofexpression in primary adipocytes of hRUP25 and hRUP38, respectively (thesignal toward the left of each of the histograms corresponding toprimary adipocytes is identified by a vertical arrow above the bar, forease of reference).

FIG. 13C is a photograph of an ethidium bromide stained gel illustratingthe relative expression of hRUP25 and hRUP38 as detected by RT-PCR usingcDNA derived from a number of human tissues as template. Note thecontrols of the far right three lanes.

FIGS. 14A-C. FIGS. 14A, 14B and 14C depict melanophores transfected withDNA plasmids expressing hRUP25 (FIG. 14A), hRUP38 (FIG. 14B) and hRUP19(FIG. 14C) without treatment. These cells are pigment-aggregated becausehRUP25 (FIG. 14A), hRUP38 (FIG. 14B) and hRUP19 (FIG. 14C) areGi-coupled receptors having a high basal level of activity, andtherefore driving the aggregation to a measurable level in the absenceof a ligand. hRUP11 is also a Gi-coupled receptor having a high basallevel of activity (not shown).

FIGS. 15A-B. FIGS. 15A and 15B illustrate the dose-dependant, nicotinicacid induced aggregation response of melanophores transfected withincreasing amounts of plasmid DNA encoding hRUP25 (FIG. 15A). Cellstransfected with 10 μg of plasmid DNA encoding hRUP25, respond tonicotinic acid with an EC₅₀ of about 54 nM.

As negative controls, FIG. 15B depicts melanophores transfected witheither salmon sperm DNA (Mock) or plasmid DNA encoding the α_(2A)AR. Asis evident there is no aggregation response in these cells uponnicotinic acid treatment at doses up to 10 μM.

FIG. 16. FIG. 16 illustrates the nicotinic acid induced-inositolphosphates (IPs) accumulation in HEK293 cells co-expressing hRUP25 andthe chimeric Gαq-subunit in which the last five amino acids have beenreplaced with the corresponding amino acids of Gαi (GqΔGi). Thisconstruct has been shown to convert the signaling of a Gi-coupledreceptor to the Gq pathway (i.e. accumulation of inositol phosphates) inresponse to receptor activation. Cells transfected with GqΔGi pluseither empty plasmid or the constitutively activated α_(2A)AR (α_(2A)K)are non-responsive to nicotinic acid and served as controls for the IPassay. Cells transfected with GqΔGi plus either hRUP19 or hRUP38 arealso unresponsive to nicotinic acid, indicating that nicotinic acid isnot an agonist for either hRUP19 or hRUP38.

FIG. 17. FIG. 17 shows the results from saturation binding of[³H]nicotinic acid to membranes from cells expressing either hRUP25,hRUP38, hRUP19 or vector alone [CHO(−)]. Note that only hRUP25 bindsnicotinic acid in a specific and high-affinity manner.

FIGS. 18A-B. FIG. 18A is a set of immunofluorescent photomicrographsillustrating the expression of hemaglutinin (HA)-tagged hRUP25 in astably transfected line of CHO cells (top; clone #46). No significantlabeling is detected in mock stably-transfected CHO cells (Mock). Thelower panels identify the nuclear (DAPI) staining of cells in the samefield.

FIG. 18B illustrates nicotinic acid and (−)-nicotine induced-inhibitionof forskolin stimulated cAMP accumulation in hRUP25-CHO cell stable line#46 (described in preceding paragraph). The EC₅₀ for nicotinic acid is23.6 nM and that for (−)-nicotine is 9.8 μM.

FIG. 19. FIG. 19 indicates that, in response to nicotinic acid, bothhRUP25 and the mouse ortholog mRUP25 can inhibit TSHR stimulated cAMPproduction (in the presence and absence of TSH).

FIG. 20. FIG. 20 shows the saturation binding curves of [³H]nicotinicacid ([³H]NA) to membranes prepared from HEK293 cells transientlyexpressing either hRUP25 or mRUP25. Note the significant binding of[³H]NA relative to either that found in membranes derived from mocktransfected cells or in the presence of an excess of non-labelednicotinic acid (200 μM).

FIG. 21. FIG. 21 is a table comparing the rank order of potency ofvarious compounds on hRUP25 and the pharmacologically defined nicotinicacid receptor. The potencies at hRUP25 derived both by a functionalanalysis measuring the inhibition of forskolin induced cAMP productionand competitive radioligand binding assays, closely match the order ofpotencies of the pharmacologically defined nicotinic acid receptor.

FIGS. 22A-B. FIG. 22A depicts nicotinic acid and related compoundsinhibiting isoproterenol induced lipolysis in rat epididymal fat derivedadipocytes at a concentration of 10 μM. P-3-T represents3-tetrazole-5-pyridine.

FIG. 22B illustrates a nicotinic acid dose-dependent inhibition ofisoproterenol induced-lipolysis in rat epididymal fat derivedadipocytes. Note the rightward shift in the dose-response curves withincreasing concentrations of nicotinic acid.

FIG. 23. FIG. 23 illustrates the ability of both nicotinic acid and therelated compound P-3-T (3-tetrazole-5-pyridine) to inhibit isoproterenolinduced lipolysis in adipocyte primary cultures derived from humansubcutaneous fat in a dose-dependant manner. The EC₅₀ value fornicotinic acid and P-3-T were 716 nM and 218 nM respectively.

FIG. 24. FIG. 24 presents screening data via adenylyl cyclase assay forhRUP38. Note that nicotinic acid does not activate inhibition offorskolin stimulated cAMP in hRUP38-expressing CHO cells whereas1-Isopropyl-1H-benzotriazole-5-carboxylic acid does.1-Isopropyl-1H-benzotriazole-5-carboxylic acid has no effect on CHOcells expressing either hRUP25 or hRUP19. The EC₅₀ for nicotinic acid is25.8 nM and that for 1-Isopropyl-1H-benzotriazole-5-carboxylic acid is166 nM. NT indicates not tested. (Also see the legend to FIG. 18A abovefor details directed to stable CHO transfectants.) Also see Example 30,infra.

FIG. 25. Nicotinic acid and 1-Isopropyl-1H-benzotriazole-5-carboxylicacid were separately dose-dependently applied to isoproterenolstimulated (100 nM) primary human adipocytes. FIG. 25 illustrates theability of 1-Isopropyl-1H-benzotriazole-5-carboxylic acid to inhibitisoproterenol stimulated lipolysis in adipocyte primary cultures derivedfrom human subcutaneous fat in a dose-dependant manner comparable tothat of nicotinic acid.

FIG. 26. FIG. 26 presents screening data via adenylyl cyclase assay forhRUP38. The horizontal axis indicates the concentration of3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid. The vertical axis indicates “%inhibition of cAMP”. Note that a value of 100% on the vertical axiscorresponds to the cAMP level of forskolin stimulated cells in theabsence of 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid, whereas a value of200% on the vertical axis corresponds to the cAMP level of unstimulatedcells in the absence of 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid. Notethat 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid activates inhibition offorskolin stimulated cAMP in hRUP38-expressing CHO cells but has noeffect on CHO cells expressing either hRUP25 or hRUP19. The EC₅₀ for3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid is 1.17 μM. (Also see thelegend to FIG. 18A above for details directed to stable CHOtransfectants.)

FIG. 27. FIG. 27 presents an RT-PCR analysis of hRUP19 expression usinga panel of human tissues. The analysis indicates that hRUP19 isselectively expressed in fat cells. Low expression is also evident intestis, placenta, kidney and spleen.

FIG. 28. FIG. 28 presents a Northern blot analysis of hRUP19 expressionusing a panel of human tissues. The analysis indicates that hRUP19 isstrongly expressed in mammary gland, probably due to fat cell-specificexpression of hRUP19. Ad, adrenal gland; Bl, bladder, BM, bone marrow;Br, brain (whole); LN, lymph node; MG, mammary gland; Pr, prostate; Sp,spinal cord; St, stomach; Thyr, thyroid; Trch, trachea; Ut, uterus.

FIG. 29. FIG. 29 presents an analysis of RUP19 expression as a functionof adipocyte differentiation. RT-PCR and Northern blot analysis ofmRUP19 expression by mouse 3T3 pre-adipocytes and differentiated 3T3adipocytes was carried out. The analysis indicates that RUP19 expressionis induced during adipocyte differentiation. Pre-diff 3T3-L1, mouse 3T3pre-adipocytes; Post-diff 3T3-L1, differentiated 3T3 adipocytes; β-TC-6,a mouse insulin-producing cell line; NIT-1, a mouse insulin-producingcell line.

FIG. 30. FIG. 30 presents a CART analysis of signal transduction byhRUP19. The analysis indicates that CART-activated hRUP19 inhibits cAMPproduction in membranes of transfected 293 cells.

FIG. 31. FIG. 31 presents screening data via adenylyl cyclase assay forhRUP25. The horizontal axis indicates the concentration of(5-hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanone.The vertical axis indicates “% inhibition of cAMP”. Note that a value of100% on the vertical axis corresponds to the cAMP level of forskolinstimulated cells in the absence of(5-hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanone,whereas a value of 200% on the vertical axis corresponds to the cAMPlevel of unstimulated cells in the absence of(5-hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanone.Note that(5-hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanoneactivates inhibition of forskolin stimulated cAMP in hRUP25-expressingCHO cells and has an EC₅₀ of 352 nM.(5-Hydroxy-1-methyl-3-propyl-1H-pyrazol-4-yl)-pyridin-3-yl-methanone hasno activity on hRUP38-expressing CHO cells up to a concentration of atleast 100 μM (not shown). Also see Example 29, infra.

FIG. 32. FIG. 32 presents a time-course analysis of plasma free fattyacids (FFA) concentration in rats administered either vehicle or niacin[NA] at 15 mg/kg, 30 mg/kg, or 45 mg/kg. Also see Example 31, infra.

FIG. 33. FIG. 33 presents an analysis of the agonist screening windowfor EFA-hRUP25 GPCR polypeptide of SEQ. ID. NO.:159 [“hRUP25-S91”]relative to that for endogenous hRUP25 GPCR polypeptide of SEQ. ID.NO.:36 [“hRUP25 wt”]. Samples were set up in triplicate. HEK293 cellswere transfected with pCMV vector alone [“CMV”], with TSHR alone[“CMV+TSHR”], or were co-transfected with TSHR and either a2AK (aconstitutively activated lysine mutant of alpha2A adrenergic receptor)[“a2AK”] or endogenous hRUP25 [“hRUP25 wt”] or EFA-hRUP25[“hRUP25-S91”]. Niacin [“Ni”] was taken as a known agonist of hRUP25.UK14,304 [“UK”] was taken as a known agonist of a2AK, a positive controlfor the assay. The level of intracellular cAMP was determined for eachsample. Also see Example 32, infra, for more details.

DETAILED DESCRIPTION

Definitions

The scientific literature that has evolved around receptors has adopteda number of terms to refer to ligands having various effects onreceptors. For clarity and consistency, the following definitions willbe used throughout this patent document. To the extent that thesedefinitions conflict with other definitions for these terms, thefollowing definitions shall control:

ADIPONECTIN. ADIPONECTIN is a plasma protein secreted by adipocytes andcomprised of an N-terminally disposed collagen-like region and aC-terminal globular region. Reduced levels of plasma ADIPONECTIN havebeen associated with a number of metabolic-related disorders, includingatherosclerosis, coronary heart disease, stroke, insulin resistance andtype 2 diabetes. The serum ADIPONECTIN level for women has been reportedto be higher than that for men, for example 13.5 μg ml⁻¹ versus 7.2 μgml⁻¹ in one study [Yamamoto Y et al., Clin Sci (Lond) (2002) 103:13742;the disclosure of which is hereby incorporated by reference in itsentirety].

AFFINITY REAGENTS shall mean compounds that specifically and measurablybind to a target molecule. Preferably the target molecule is a GPCR.

AGONISTS shall mean materials (e.g., ligands, candidate compounds) thatactivate an intracellular response when they bind to the receptor. Insome embodiments, AGONISTS are those materials not previously known toactivate the intracellular response when they bind to the receptor (e.g.to enhance GTPγS binding to membranes or to lower intracellular cAMPlevel). In some embodiments, AGONISTS are those materials not previouslyknown to inhibit lipolysis when they bind to the receptor.

ALLELIC VARIANT. See VARIANT.

ALLOSTERIC MODULATORS shall mean materials (e.g., ligands, candidatecompounds) that affect the functional activity of the receptor but whichdo not inhibit the endogenous ligand from binding to the receptor.Allosteric modulators include inverse agonists, partial agonists andagonists.

AMINO ACID ABBREVIATIONS used herein are set out in Table A: TABLE AALANINE ALA A ARGININE ARG R ASPARAGINE ASN N ASPARTIC ACID ASP DCYSTEINE CYS C GLUTAMIC ACID GLU E GLUTAMINE GLN Q GLYCINE GLY GHISTIDINE HIS H ISOLEUCINE ILE I LEUCINE LEU L LYSINE LYS K METHIONINEMET M PHENYLALANINE PHE F PROLINE PRO P SERINE SER S THREONINE THR TTRYPTOPHAN TRP W TYROSINE TYR Y VALINE VAL V

ANTAGONISTS shall mean materials (e.g., ligands, candidate compounds)that competitively bind to the receptor at the same site as the agonistsbut which do not activate an intracellular response, and can therebyinhibit the intracellular responses elicited by agonists. ANTAGONISTS donot diminish the baseline intracellular response in the absence of anagonist. In some embodiments, ANTAGONISTS are those materials notpreviously known to compete with an agonist to inhibit the cellularresponse when they bind to the receptor, e.g. wherein the cellularresponse is GTPγS binding to membranes or to the lowering ofintracellular cAMP level.

ANTIBODIES are intended herein to encompass monoclonal antibodies andpolyclonal antibodies. ANTIBODIES are further intended to encompass IgG,IgA, IgD, IgE, and IgM. ANTIBODIES include whole antibodies, includingsingle-chain whole antibodies, and antigen binding fragments thereof,including Fab, Fab′, F(ab)2 and F(ab′)2. ANTIBODIES may be from anyanimal origin. Preferably, ANTIBODIES are human, murine, rabbit, goat,guinea pig, hamster, camel, donkey, sheep, horse or chicken. PreferablyANTIBODIES have binding affinities with a dissociation constant or Kdvalue less than 5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M,5×10⁻⁹ M,

10⁻⁹ M, 5×10⁻¹⁰ M 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M,5×10⁻¹³ M, 10⁻¹³ M,

5×10⁻¹⁴ M 10⁻¹⁴ M, 5×10⁻¹⁵ M and 10⁻¹⁵ M. ANTIBODIES of the presentinvention may be prepared by any suitable method known in the art.

ATHEROSCLEROSIS is intended herein to encompass disorders of large andmedium-sized arteries that result in the progressive accumulation withinthe intima of smooth muscle cells and lipids. Atherosclerosis is theprimary cause of heart disease and stroke.

BIOLOGICALLY ACTIVE FRAGMENT is interchangeable herein with ACTIVEFRAGMENT and shall mean a fragment of full-length polypeptide orfull-length amino acid sequence retaining part or all of thefunctionality of said full-length polypeptide or full-length amino acidsequence. In particular embodiment, a GPCR comprising an active fragmentof a full-length GPCR polypeptide or full-length GPCR amino acidsequence retains part or all of the functionality of said GPCRcomprising said full-length polypeptide or said full-length amino acidsequence. Said GPCR functionality is understood to include but notintended to be limited to ligand binding, G protein coupling, andligand-facilitated coupling to G protein. By way of illustration and notlimitation, BIOLOGICALLY ACTIVE FRAGMENT is intended herein to encompassfull-length GPCR polypeptide absent the N-terminal methionine.

3-(5-BROMO-2-ETHOXY-PHENYL)-ACRYLIC ACID shall be understood herein tohave the formula:

and to encompass the E isomer, the Z isomer, and mixtures of E and Zisomers.

CANDIDATE COMPOUND shall mean a molecule (for example, and notlimitation, a chemical compound) that is amenable to a screeningtechnique. Preferably, the phrase “candidate compound” does not includecompounds which were publicly known to be compounds selected from thegroup consisting of inverse agonist, agonist or antagonist to areceptor, as previously determined by an indirect identification process(“indirectly identified compound”); more preferably, not including anindirectly identified compound which has previously been determined tohave therapeutic efficacy in at least one mammal; and, most preferably,not including an indirectly identified compound which has previouslybeen determined to have therapeutic utility in humans.

CHOLESTEROL. Generally, the total cholesterol/HDL-cholesterol (i.e.,TC/HDL) ratio represents a useful predictor as to the risk of anindividual in developing a more serious condition, such as anHDL-related condition, such as but not limited to atherosclerosis andcomplications therefrom. The classification of plasma lipid levels isshown in Chart A: CHART A CLASSIFICATION OF PLASMA LIPID LEVELS TOTAL<200 mg/dl Desirable CHOLESTEROL 200-239 mg/dl Borderline High >240mg/dl High HDL- <40 mg/dl Low (Men) CHOLESTEROL <50 mg/dl Low(Women) >60 mg/dl HighFrom: 2001 National Cholesterol Education Program GuidelinesAccordingly, the recommended total cholesterol/HDL-C (i.e., TC/HDL)ratio indicates that a ratio of less than or equal to 3.5 is ideal and aratio of greater than 4.5 is considered an increased “at risk.” Thevalue of determining the TC/HDL ratio is clearly evident in thecircumstance where an individual presents with “normal” LDL and totalcholesterol but possesses low HDL-cholesterol. Based on LDL and totalcholesterol the individual may not qualify for treatment however, factorin the HDL-cholesterol level then a more accurate risk assessment may beobtained. Thus, if the individual's level of HDL-cholesterol is suchthat the ratio is greater than 4.5 then therapeutic or preventiveintervention may be warranted. A physician or care provider maydetermine the need of prevention or treatment based on a TC/HDL ratio;for example, a TC/HDL ratio of 2.5 or greater, 3.0 or greater, 3.5 orgreater, 4.0 or greater, 4.5 or greater, 5.0 or greater, or a TC/HDLratio of 5.5 or greater.

CODON shall mean a grouping of three nucleotides (or equivalents tonucleotides) which generally comprise a nucleoside [adenosine (A),guanosine (G), cytidine (C), uridine (U) and thymidine (T)] coupled to aphosphate group and which, when translated, encodes an amino acid.

COMPOSITION means a material comprising at least one component; a“pharmaceutical composition” is an example of a composition.

COMPOUND EFFICACY shall mean a measurement of the ability of a compoundto inhibit or stimulate receptor functionality; i.e. the ability toactivate/inhibit a signal transduction pathway, in contrast to receptorbinding affinity. Exemplary means of detecting compound efficacy aredisclosed in the Example section of this patent document.

COMPRISING, CONSISTING ESSENTIALLY OF, and CONSISTING OF are definedherein according to their standard meaning. A defined meaning set forthin the M.P.E.P. controls over a defined meaning in the art and a definedmeaning set forth in controlling Federal Circuit case law controls overa meaning set forth in the M.P.E.P.

CONSTITUTIVELY ACTIVE RECEPTOR shall mean a receptor stabilized in anactive state by means other than through binding of the receptor to itsligand or a chemical equivalent thereof. A CONSTITUTIVELY ACTIVERECEPTOR may be endogenous or non-endogenous.

CONSTITUTIVELY ACTIVATED RECEPTOR shall mean an endogenous receptor thathas been modified so as to be constitutively active. CART is an acronymfor Constitutively Activated Receptor Technology and when used hereinprefixing or suffixing a GPCR, shall be understood to identify saidprefixed or suffixed GPCR as a CONSTITUTIVELY ACTIVATED RECEPTOR.

CONSTITUTIVE RECEPTOR ACTIVATION shall mean activation of a receptor inthe absence of binding to its ligand or a chemical equivalent thereof.

CONTACT or CONTACTING shall mean bringing at least two moietiestogether, whether in an in vitro system or an in vivo system.

CORONARY HEART DISEASE is intended herein to encompass disorderscomprising a narrowing of the small blood vessels that supply blood andoxygen to the heart. CORONARY HEART DISEASE usually results from thebuild up of fatty material and plaque. As the coronary arteries narrow,the flow of blood to the heart can slow or stop. CORONARY HEART DISEASEcan cause chest pain (stable angina), shortness of breath, heart attack,or other symptoms. CORONARY HEART DISEASE is intended herein to includecoronary artery disease, the most common type of heart disease. Coronaryartery disease results from atherosclerosis.

DECREASE is used to refer to a reduction in a measurable quantity and isused synonymously with the terms “reduce”, “diminish”, “lower”, and“lessen”.

DIABETES as used herein is intended to encompass the usual diagnosis ofDIABETES made from any of the methods including, but not limited to, thefollowing list: symptoms of diabetes (e.g., polyuria, polydipsia,polyphagia) plus casual plasma glucose levels of greater than or equalto 200 mg/dl, wherein casual plasma glucose is defined any time of theday regardless of the timing of meal or drink consumption; 8 hourfasting plasma glucose levels of less than or equal to 126 mg/dl; andplasma glucose levels of greater than or equal to 200 mg/dl 2 hoursfollowing oral administration of 75 g anhydrous glucose dissolved inwater.

DIRECTLY IDENTIFYING or DIRECTLY IDENTIFIED, in relationship to thephrase “candidate compound”, shall mean the screening of a candidatecompound against a constitutively activated receptor, preferably aconstitutively activated orphan receptor, and most preferably against aconstitutively activated G protein-coupled cell surface orphan receptor,and assessing the compound efficacy of such compound. This phrase is,under no circumstances, to be interpreted or understood to beencompassed by or to encompass the phrase “indirectly identifying” or“indirectly identified.”

DISORDERS OF LIPID METABOLISM are intended herein to include, but not belimited to, dyslipidemia.

DYSLIPIDEMIA is intended herein to encompass disorders comprising anyone of elevated level of plasma free fatty acids, elevated level ofplasma cholesterol, elevated level of LDL-cholesterol, reduced level ofHDL-cholesterol, elevated ratio of total cholesterol to HDL-cholesterol,and elevated level of plasma triglycerides.

EFA-GPCR shall mean a mutant GPCR polypeptide that consists of 1, 2, 3,4, or 5 amino acid substitutions, deletions, or insertions relative tothe amino acid sequence of an endogenous GPCR polypeptide havingconstitutive activity, wherein the agonist screening window of themutant GPCR is expanded by greater than 20%, greater than 25%, greaterthan 30%, greater than 31%, greater than 32%, greater than 33%, greaterthan 34%, greater than 35%, greater than 36%, greater than 37%, greaterthan 38%, greater than 39%, or greater than 40% relative to that of saidendogenous GPCR.

ENDOGENOUS shall mean a material that a mammal naturally produces.ENDOGENOUS in reference to, for example and not limitation, the term“receptor,” shall mean that which is naturally produced by a mammal (forexample, and not limitation, a human) or a virus. ENDOGENOUS shall beunderstood to encompass allelic variants of a gene as well as theallelic polypeptide variants so encoded. By contrast, the termNON-ENDOGENOUS in this context shall mean that which is not naturallyproduced by a mammal (for example, and not limitation, a human) or avirus. For example, and not limitation, a receptor which is notconstitutively active in its endogenous form, but when manipulatedbecomes constitutively active, is most preferably referred to herein asa “non-endogenous, constitutively activated receptor.” Both terms can beutilized to describe both “in vivo” and “in vitro” systems. For example,and not limitation, in a screening approach, the endogenous ornon-endogenous receptor may be in reference to an in vitro screeningsystem. As a further example and not limitation, where the genome of amammal has been manipulated to include a non-endogenous constitutivelyactivated receptor, screening of a candidate compound by means of an invivo system is viable.

EXPRESSION VECTOR is defined herein as a DNA sequence that is requiredfor the transcription of cloned DNA and the translation of thetranscribed mRNAs in an appropriate host cell recombinant for saidEXPRESSION VECTOR. An appropriately constructed EXPRESSION VECTOR shouldcontain an origin of replication for autonomous replication in hostcells, selectable markers, a limited number of useful restriction enzymesites, a potential for high copy number, and active promoters. By way ofillustration and not limitation, pCMV is an expression vector.

G PROTEIN COUPLED RECEPTOR FUSION PROTEIN and GPCR FUSION PROTEIN, inthe context of the invention disclosed herein, each mean anon-endogenous protein comprising an endogenous, constitutively activateGPCR or a non-endogenous, constitutively activated GPCR fused to atleast one G protein, most preferably the alpha (a) subunit of such Gprotein (this being the subunit that binds GTP), with the G proteinpreferably being of the same type as the G protein that naturallycouples with endogenous orphan GPCR. For example, and not limitation, inan endogenous state, if the G protein “G_(s)α” is the predominate Gprotein that couples with the GPCR, a GPCR Fusion Protein based upon thespecific GPCR would be a non-endogenous protein comprising the GPCRfused to G_(s)α; in some circumstances, as will be set forth below, anon-predominant G protein can be fused to the GPCR. The G protein can befused directly to the C-terminus of the constitutively active GPCR orthere may be spacers between the two.

HOST CELL shall mean a cell capable of having a Plasmid and/or Vectorincorporated therein. In the case of a prokaryotic Host Cell, a Plasmidis typically replicated as a autonomous molecule as the Host Cellreplicates (generally, the Plasmid is thereafter isolated forintroduction into a eukaryotic Host Cell); in the case of a eukaryoticHost Cell, a Plasmid may be integrated into the cellular DNA of the HostCell such that when the eukaryotic Host Cell replicates, the Plasmidreplicates. In some embodiments the Host Cell is eukaryotic, morepreferably, mammalian, and more preferably selected from the groupconsisting of 293, 293T, CHO, and COS-7 cells. In other embodiments, theHost Cell is eukaryotic, more preferably melanophore.

(5-HYDROXY-1-METHYL-3-PROPYL-1H-PYRAZOL-4-YL)-PYRIDIN-3-YL-METHANONEshall be understood herein to have the formula:

IN NEED OF PREVENTION OR TREATMENT as used herein refers to a judgementmade by a caregiver (e.g. physician, nurse, nurse practitioner, etc. inthe case of humans; veterinarian in the case of animals, includingnon-human mammals) that an individual or animal requires or will benefitfrom treatment. This judgement is made based on a variety of factorsthat are in the realm of a caregiver's expertise, but that include theknowledge that the individual or animal is ill, or will be ill, as theresult of a condition that is treatable by the compounds of theinvention.

INDIRECTLY IDENTIFYING or INDIRECTLY IDENTIFIED means the traditionalapproach to the drug discovery process involving identification of anendogenous ligand specific for an endogenous receptor, screening ofcandidate compounds against the receptor for determination of thosewhich interfere and/or compete with the ligand-receptor interaction, andassessing the efficacy of the compound for affecting at least one secondmessenger pathway associated with the activated receptor.

INDIVIDUAL as used herein refers to any animal, including mammals,preferably mice, rats, other rodents, rabbits, dogs, cats, swine,cattle, sheep, horses, or primates, and most preferably humans.

INHIBIT or INHIBITING, in relationship to the term “response” shall meanthat a response is decreased or prevented in the presence of a compoundas opposed to in the absence of the compound.

INSULIN RESISTANCE as used herein is intended to encompass the usualdiagnosis of insulin resistance made by any of a number of methods,including but not restricted to: the intravenous glucose tolerance testor measurement of the fasting insulin level. It is well known that thereis an excellent correlation between the height of the fasting insulinlevel and the degree of insulin resistance. Therefore, one could useelevated fasting insulin levels as a surrogate marker for insulinresistance for the purpose of identifying which normal glucose tolerance(NGT) individuals have insulin resistance. A diagnosis of insulinresistance can also be made using the euglycemic glucose clamp test.

INVERSE AGONISTS shall mean materials (e.g., ligand, candidate compound)that bind either to the endogenous form or to the constitutivelyactivated form of the receptor so as to reduce the baselineintracellular response of the receptor observed in the absence ofagonists.

ISOLATED shall mean that the material is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or DNA or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Such apolynucleotide could be part of a vector and/or such a polynucleotide orpolypeptide could be part of a composition, and still be isolated inthat the vector or composition is not part of its natural environment.

1-ISOPROPYL-1H-BENZOTRIAZOLE-5-CARBOXYLIC ACID shall be understoodherein to have the formula:

KNOCKOUT MOUSE/RAT is intended herein to encompass a mouse or rat thathas been manipulated by recombinant means such that a single gene ofchoice has been inactivated or “knocked-out” in a manner that leaves allother genes unaffected.

KNOWN RECEPTOR shall mean an endogenous receptor for which theendogenous ligand specific for that receptor has been identified.

LIGAND shall mean a molecule specific for a naturally occurringreceptor.

METABOLIC-RELATED DISORDERS are intended herein to include, but not belimited to, dyslipidemia, atherosclerosis, coronary heart disease,stroke, insulin resistance and type 2 diabetes.

As used herein, the terms MODULATE or MODIFY are meant to refer to anincrease or decrease in the amount, quality, or effect of a particularactivity, function or molecule.

MUTANT or MUTATION in reference to an endogenous receptor's nucleic acidand/or amino acid sequence shall mean a specified change or changes tosuch endogenous sequences such that a mutated form of an endogenousnon-constitutively activated receptor evidences constitutive activationof the receptor. In terms of equivalents to specific sequences, asubsequent mutated form of a human receptor is considered to beequivalent to a first mutation of the human receptor if (a) the level ofconstitutive activation of the subsequent mutated form of a humanreceptor is substantially the same as that evidenced by the firstmutation of the receptor; and (b) the percent sequence (amino acidand/or nucleic acid) homology between the subsequent mutated form of thereceptor and the first mutation of the receptor is at least 80%, atleast 85%, at least 90%, at least 92%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, and most preferably at least 99%.In some embodiments, owing to the fact that some preferred cassettesdisclosed herein for achieving constitutive activation include a singleamino acid and/or codon change between the endogenous and thenon-endogenous forms of the GPCR, it is preferred that the percentsequence homology should be at least 98%.

(−)-NICOTINE shall be understood herein to have the formula:

NICOTINIC ACID shall be understood herein to have the formula:

As used herein, the term NICOTINIC ACID ANALOG OR DERIVATIVE is meant tomolecules which bind to nicotinic acid receptors and have substantiallysimilar effects on the receptor. Such analogs and derivatives arewell-known to those skilled in the art and include, but are not limitedto, Acipimox™ and niacinamide.

NON-ORPHAN RECEPTOR shall mean an endogenous naturally occurringmolecule specific for an identified ligand wherein the binding of aligand to a receptor activates an intracellular signaling pathway.

ORPHAN RECEPTOR shall mean an endogenous receptor for which the ligandspecific for that receptor has not been identified or is not known.

PARTIAL AGONISTS shall mean materials (e.g., ligands, candidatecompounds) that activate the intracellular response when they bind tothe receptor to a lesser degree/extent than do full agonists.

PHARMACEUTICAL COMPOSITION shall mean a composition comprising at leastone active ingredient, whereby the composition is amenable toinvestigation for a specified, efficacious outcome in a mammal (forexample, and not limitation, a human). Those of ordinary skill in theart will understand and appreciate the techniques appropriate fordetermining whether an active ingredient has a desired efficaciousoutcome based upon the needs of the artisan.

PLASMID shall mean the combination of a Vector and cDNA. Generally, aPlasmid is introduced into a Host Cell for the purposes of replicationand/or expression of the cDNA as a protein.

POLYNUCLEOTIDES shall mean RNA, DNA, or RNA/DNA hybrid sequences of morethan one nucleotide in either single chain or duplex form. Thepolynucleotides of the invention may be prepared by any known method,including synthetic, recombinant, ex vivo generation, or a combinationthereof, as well as utilizing any purification methods known in the art.

POLYPEPTIDE shall refer to a polymer of amino acids without regard tothe length of the polymer. Thus, peptides, oligopeptides, and proteinsare included within the definition of polypeptide. This term also doesnot specify or exclude post-expression modifications of polypeptides.For example, polypeptides that include the covalent attachment ofglycosyl groups, acetyl groups, phosphate groups, lipid groups and thelike are expressly encompassed by the term POLYPEPTIDE.

PRIMER is used herein to denote a specific oligonucleotide sequencewhich is complementary to a target nucleotide sequence and used tohybridize to the target nucleotide sequence. A primer serves as aninitiation point for nucleotide polymerization catalyzed by DNApolymerase, RNA polymerase, or reverse transcriptase.

PURIFIED is used herein to describe a polynucleotide or polynucleotidevector of the invention that has been separated from other compoundsincluding, but not limited to, other nucleic acids, carbohydrates,lipids and proteins (such as the enzymes used in the synthesis of thepolynucleotide). A polynucleotide is substantially pure when at leastabout 50%, 60%, 75%, or 90% of a sample contains a single polynucleotidesequence. A substantially pure polynucleotide typically comprises about50, 60, 70, 80, 90, 95, 99% weight/weight of a nucleic acid sample.Polynucleotide purity or homogeneity may be indicated by a number ofmeans well known in the art, such as agarose or polyacrylamide gelelectrophoresis of a sample, followed by visualizing a singlepolynucleotide band upon staining the gel.

Similarly, the term PURIFIED is used herein to describe a polypeptide ofthe invention that has been separated from other compounds including,but not limited to, nucleic acids, lipids, carbohydrates and otherproteins. In some preferred embodiments, a polypeptide is substantiallypure when at least about 50%, 60%, 75%, 85%, 90%, 95%, 96%, 97%, 98%,99%, or 99.5% of the polypeptide molecules of a sample have a singleamino acid sequence. In some preferred embodiments, a substantially purepolypeptide typically comprises about 50%, 60%, 70%, 80%, 90%, 95%, 96%,97%, 98%, 99% or 99.5% weight/weight of a protein sample. Polypeptidepurity or homogeneity is indicated by a number of methods well known inthe art, such as agarose or polyacrylamide gel electrophoresis of asample, followed by visualizing a single polypeptide band upon stainingthe gel.

Further, as used herein, the term PURIFIED does not require absolutepurity; rather, it is intended as a relative definition. Purification ofstarting material or natural material to at least one order ofmagnitude, preferably two or three orders, and more preferably four orfive orders of magnitude is expressly contemplated.

RECEPTOR FUNCTIONALITY shall refer to the normal operation of a

receptor to receive a stimulus and moderate an effect in the cell,including, but not limited to regulating gene transcription, regulatingthe influx or efflux of ions, effecting a catalytic reaction, and/ormodulating activity through G-proteins.

SECOND MESSENGER shall mean an intracellular response produced as aresult of receptor activation. A second messenger can include, forexample, inositol triphosphate (IP₃), diacylglycerol (DAG), cyclic AMP(cAMP), cyclic GMP (cGMP), and Ca²⁺. Second messenger response can bemeasured for a determination of receptor activation. In addition, secondmessenger response can be measured for the direct identification ofcandidate compounds, including for example, inverse agonists, partialagonists, agonists, and antagonists.

SIGNAL TO NOISE RATIO shall mean the signal generated in response toactivation, amplification, or stimulation wherein the signal is abovethe background noise or the basal level in response to non-activation,non-amplification, or non-stimulation.

SPACER shall mean a translated number of amino acids that are locatedafter the last codon or last amino acid of a gene, for example a GPCR ofinterest, but before the start codon or beginning regions of the Gprotein of interest, wherein the translated number amino acids areplaced in-frame with the beginnings regions of the G protein ofinterest. The number of translated amino acids can be one, two, three,four, etc., and up to twelve.

STIMULATE or STIMULATING, in relationship to the term “response” shallmean that a response is increased in the presence of a compound asopposed to in the absence of the compound.

STROKE is a cardiovascular disease that affects the blood vesselssupplying blood to the brain and is intended herein to include cerebralthrombosis, the most common type of STROKE. Cerebral thrombosis occurswhen a blood clot (thrombus) forms and blocks blood flow in an arterybringing blood to part of the brain. Blood clots usually form inarteries damaged by atherosclerosis.

SUBJECT shall mean primates, including but not limited to humans andbaboons, as well as pet animals such as dogs and cats, laboratoryanimals such as rats and mice, and farm animals such as horses, sheep,and cows.

SUBSTANTIALLY shall refer to a result which is within 40% of a controlresult, preferably within 35%, more preferably within 30%, morepreferably within 25%, more preferably within 20%, more preferablywithin 15%, more preferably within 10%, more preferably within 5%, morepreferably within 2%, and most preferably within 1% of a control result.For example, in the context of receptor functionality, a test receptormay exhibit substantially similar results to a control receptor if thetransduced signal, measured using a method taught herein or similarmethod known to the art-skilled, is within 40% of the signal produced bya control signal.

TRANSGENIC MOUSE/RAT shall be intended herein to encompass a mouse orrat that has been engineered through recombinant means to carry aforeign gene, or transgene, of choice as part of its own geneticmaterial.

VARIANT as the term is used herein, is a polynucleotide or polypeptidethat differs from a reference polynucleotide or polypeptiderespectively, but retains essential properties. A typical variant of apolynucleotide differs in nucleotide sequence from another, referencepolynucleotide. Changes in the nucleotide sequence of the variant may ormay not alter the amino acid sequence of a polypeptide encoded by thereference polynucleotide. A typical variant of a polypeptide differs inamino acid sequence from another, reference polypeptide. A variant andreference polypeptide may differ in amino acid sequence by one or moresubstitutions, additions, deletions in any combination. A variant of apolynucleotide or polypeptide may be a naturally occurring one such asan ALLELIC VARIANT, or it may be a variant that is not known to occurnaturally. Non-naturally occurring variants of polynucleotides andpolypeptides may be made by mutagenesis techniques or by directsynthesis.

VECTOR in reference to cDNA shall mean a circular DNA capable ofincorporating at least one cDNA and capable of incorporation into a HostCell.

The order of the following sections is set forth for presentationalefficiency and is not intended, nor should be construed, as a limitationon the disclosure or the claims to follow.

A. Introduction

The traditional study of receptors has always proceeded from the apriori assumption (historically based) that the endogenous ligand mustfirst be identified before discovery could proceed to find antagonistsand other molecules that could affect the receptor. Even in cases wherean antagonist might have been known first, the search immediatelyextended to looking for the endogenous ligand. This mode of thinking haspersisted in receptor research even after the discovery ofconstitutively activated receptors. What has not been heretoforerecognized is that it is the active state of the receptor that is mostuseful for discovering agonists, partial agonists, and inverse agonistsof the receptor. For those diseases which result from an overly activereceptor or an under-active receptor, what is desired in a therapeuticdrug is a compound which acts to diminish the active state of a receptoror enhance the activity of the receptor, respectively, not necessarily adrug which is an antagonist to the endogenous ligand. This is because acompound that reduces or enhances the activity of the active receptorstate need not bind at the same site as the endogenous ligand. Thus, astaught by the present invention, in some preferred embodiments, a searchfor therapeutic compounds should start by screening compounds againstthe ligand-independent active state.

B. Identification of Human GPCRs

The efforts of the Human Genome project has led to the identification ofa plethora of information regarding nucleic acid sequences locatedwithin the human genome; it has been the case in this endeavor thatgenetic sequence information has been made available without anunderstanding or recognition as to whether or not any particular genomicsequence does or may contain open-reading frame information thattranslate human proteins. Several methods of identifying nucleic acidsequences within the human genome are within the purview of those havingordinary skill in the art. For example, and not limitation, a variety ofhuman GPCRs, disclosed herein, were discovered by reviewing the GenBank™database. Table B, below, lists several endogenous GPCRs that we havediscovered, along with other GPCRs that are homologous to the disclosedGPCR. TABLE B Disclosed Open PerCent Human Accession Reading ReferenceTo Homology Orphan Number Frame Homologous To Designated GPCRsIdentified (Base Pairs) GPCR GPCR hRUP8 AL121755 1,152 bp NPY2R 27%hRUP9 AC0113375 1,260 bp GAL2R 22% hRUP10 AC008745 1,014 bp C5aR 40%hRUP11 AC013396 1,272 bp HM74 36% hRUP12 AP000808   966 bp Mas1 34%hRUP13 AC011780 1,356 bp Fish GPRX- 43% ORYLA hRUP14 AL137118 1,041 bpCysLT1R 35% hRUP15 AL016468 1,527 bp RE2 30% hRUP16 AL136106 1,068 bpGLR101 37% hRUP17 AC023078   969 bp Mas1 37% hRUP18 AC008547 1,305 bpOxytocin 31% hRUP19 AC026331 1,041 bp HM74 52% hRUP20 AL161458 1,011 bpGPR34 25% hRUP21 AC026756 1,014 bp P2Y1R 37% hRUP22 AC027026   993 bphRUP17 67% Mas1 37% hRUP23 AC007104 1,092 bp Rat GPR26 31% hRUP24AL355388 1,125 bp SALPR 44% hRUP25 AC026331 1,092 bp HM74 95% hRUP26AC023040 1,044 bp Rabbit 5HT1D 27% hRUP27 AC027643 1,020 bp MCH 38%hRUP38 AC026331 1,164 bp HM74 100%

Such receptors are disclosed, for example, in application Ser. No.09/714,008, filed Nov. 16, 2000, which is incorporated by reference inits entirety.

Receptor homology is useful in terms of gaining an appreciation of arole of the receptors within the human body. As the patent documentprogresses, techniques for mutating these receptors to establishnon-endogenous, constitutively activated versions of these receptorswill be discussed.

The techniques disclosed herein have also been applied to other human,orphan GPCRs known to the art, as will be apparent as the patentdocument progresses.

C. Identification of the Mouse (m) and Rat (r) Orthologs of Human (h)RUP25 and Identification of the Mouse (m) and Rat (r) Orthologs of Human(h) RUP19 TABLE C Reference Disclosed Open To PerCent Mouse (m)Accession Reading Orthologous Homology or Rat (r) Number Frame Human ToDesignated RUP25 Identified (Base Pairs) GPCR GPCR mRUP25 AJ300199 1,083bp hRUP25 83% rRUP25 None 1,086 bp hRUP25 71% mRUP19 XM_144529 1,032 bphRUP19 81% rRUP19 None 1,056 bp hRUP19 83%D. Receptor Screening

Screening candidate compounds against a non-endogenous, constitutivelyactivated version of the GPCRs disclosed herein allows for the directidentification of candidate compounds which act at the cell surfacereceptor, without requiring use, or, in some embodiments, of theknowledge of the identity of the receptor's endogenous ligand. Usingroutine and often commercially available techniques, one can determineareas within the body where the endogenous version of human GPCRsdisclosed herein is expressed and/or over-expressed. The expressionlocation of a receptor in a specific tissue provides a scientist withthe ability to assign a physiological functional role of the receptor.It is also possible using these techniques to determine relateddisease/disorder states which are associated with the expression and/orover-expression of the receptor; such an approach is disclosed in thispatent document. Furthermore, expression of a receptor in diseasedorgans can assist one in determining the magnitude of the clinicalrelevance of the receptor.

Constitutive activation of the GPCRs disclosed herein is based upon thedistance from the proline residue at which is presumed to be locatedwithin TM6 of the GPCR; this algorithmic technique is disclosed inco-pending and commonly assigned patent document PCT Application NumberPCT/US99/23938, published as WO 00/22129 on Apr. 20, 2000, which, alongwith the other patent documents listed herein, is incorporated herein byreference in its entirety. The algorithmic technique is not predicatedupon traditional sequence “alignment” but rather a specified distancefrom the aforementioned TM6 proline residue (or, of course, endogenousconstitutive substitution for such proline residue). By mutating theamino acid residue located 16 amino acid residues from this residue(presumably located in the IC3 region of the receptor) to, preferably, alysine residue, constitutive activation of the receptor may be obtained.Other amino acid residues may be useful in the mutation at this positionto achieve this objective and will be discussed in detail, below.

E. Disease/Disorder Identification and/or Selection

As will be set forth in greater detail below, inverse agonists andagonists to the non-endogenous, constitutively activated GPCR can beidentified by the methodologies of this invention. Such inverse agonistsand agonists are good candidates as lead compounds in drug discoveryprograms for treating diseases and/or disorders related to thisreceptor. Because of the ability to directly identify inverse agonistsand agonists to the GPCR, thereby allowing for the development ofpharmaceutical compositions, a search for diseases and disordersassociated with the GPCR is relevant. The expression location of areceptor in a specific tissue provides a scientist with the ability toassign a physiological function to the receptor. For example, scanningboth diseased and normal tissue samples for the presence of the GPCR nowbecomes more than an academic exercise or one which might be pursuedalong the path of identifying an endogenous ligand to the specific GPCR.Tissue scans can be conducted across a broad range of healthy anddiseased tissues. Such tissue scans provide a potential first step inassociating a specific receptor with a disease and/or disorder.Furthermore, expression of a receptor in diseased organs can assist onein determining the magnitude of the clinical relevance of the receptor.

The DNA sequence of the GPCR can be used to make a probe/primer. In somepreferred embodiments the DNA sequence is used to make a probe for (a)dot-blot analysis against tissue-mRNA, and/or (b) RT-PCR identificationof the expression of the receptor in tissue samples. The presence of areceptor in a tissue source, or a diseased tissue, or the presence ofthe receptor at elevated concentrations in diseased tissue compared to anormal tissue, can be used to correlate location to function andindicate the receptor's physiological role/function and create atreatment regimen, including but not limited to, a disease associatedwith that function/role. Receptors can also be localized to regions oforgans by this technique. Based on the known or assumed roles/functionsof the specific tissues to which the receptor is localized, the putativephysiological function of the receptor can be deduced. For example andnot limitation, proteins located/expressed in areas of the thalamus areassociated with sensorimotor processing and arousal (see, Goodman &Gilman's, The Pharmacological Basis of Therapeutics, 9^(th) Edition,page 465 (1996)). Proteins expressed in the hippocampus or in Schwanncells are associated with learning and memory, and myelination ofperipheral nerves, respectively (see, Kandel, E. et al., Essentials ofNeural Science and Behavior pages 657, 680 and 28, respectively (1995)).

F. Screening of Candidate Compounds

1. Generic GPCR Screening Assay Techniques

When a G protein receptor becomes constitutively active, it binds to a Gprotein (e.g., Gq, Gs, Gi, Gz, Go) and stimulates the binding of GTP tothe G protein. The G protein then acts as a GTPase and slowly hydrolyzesthe GTP to GDP, whereby the receptor, under normal conditions, becomesdeactivated. However, constitutively activated receptors continue toexchange GDP to GTP. A non-hydrolyzable analog of GTP, [³⁵S]GTPγS, canbe used to monitor enhanced binding to membranes which expressconstitutively activated receptors. It is reported that [³⁵S]GTPγS canbe used to monitor G protein coupling to membranes in the absence andpresence of ligand. An example of this monitoring, among other exampleswell-known and available to those in the art, was reported by Traynorand Nahorski in 1995. The preferred use of this assay system is forinitial screening of candidate compounds because the system isgenerically applicable to all G protein-coupled receptors regardless ofthe particular G protein that interacts with the intracellular domain ofthe receptor.

2. Specific GPCR Screening Assay Techniques

Once candidate compounds are identified using the “generic” Gprotein-coupled receptor assay (i.e., an assay to select compounds thatare agonists or inverse agonists), in some embodiments further screeningto confirm that the compounds have interacted at the receptor site ispreferred. For example, a compound identified by the “generic” assay maynot bind to the receptor, but may instead merely “uncouple” the Gprotein from the intracellular domain.

a. Gs, Gz and Gi.

Gs stimulates the enzyme adenylyl cyclase. Gi (and Gz and Go), on theother hand, inhibit adenylyl cyclase. Adenylyl cyclase catalyzes theconversion of ATP to cAMP; thus, constitutively activated GPCRs thatcouple the Gs protein are associated with increased cellular levels ofcAMP. On the other hand, constitutively activated GPCRs that couple Gi(or Gz, Go) protein are associated with decreased cellular levels ofcAMP. See, generally, “Indirect Mechanisms of Synaptic Transmission,”Chpt. 8, From Neuron To Brain (3^(rd) Ed.) Nichols, J. G. et al eds.Sinauer Associates, Inc. (1992). Thus, assays that detect cAMP can beutilized to determine if a candidate compound is, e.g., an inverseagonist to the receptor (i.e., such a compound would decrease the levelsof cAMP). A variety of approaches known in the art for measuring cAMPcan be utilized; in some embodiments a preferred approach relies uponthe use of anti-cAMP antibodies in an ELISA-based format. Another typeof assay that can be utilized is a whole cell second messenger reportersystem assay. Promoters on genes drive the expression of the proteinsthat a particular gene encodes. Cyclic AMP drives gene expression bypromoting the binding of a cAMP-responsive DNA binding protein ortranscription factor (CREB) that then binds to the promoter at specificsites called cAMP response elements and drives the expression of thegene. Reporter systems can be constructed which have a promotercontaining multiple cAMP response elements before the reporter gene,e.g., β-galactosidase or luciferase. Thus, a constitutively activatedGs-linked receptor causes the accumulation of cAMP that then activatesthe gene and expression of the reporter protein. The reporter proteinsuch as β-galactosidase or luciferase can then be detected usingstandard biochemical assays (Chen et al. 1995).

b. Go and Gq.

Gq and Go are associated with activation of the enzyme phospholipase C,which in turn hydrolyzes the phospholipid PIP₂, releasing twointracellular messengers: diacycloglycerol (DAG) and inistol1,4,5-triphoisphate (IP₃). Increased accumulation of IP₃ is associatedwith activation of Gq- and Go-associated receptors. See, generally,“Indirect Mechanisms of Synaptic Transmission,” Chpt. 8, From Neuron ToBrain (3^(rd) Ed.) Nichols, J. G. et al eds. Sinauer Associates, Inc.(1992). Assays that detect IP₃ accumulation can be utilized to determineif a candidate compound is, e.g., an inverse agonist to a Gq- orGo-associated receptor (i.e., such a compound would decrease the levelsof IP₃). Gq-associated receptors can also been examined using an AP 1reporter assay in that Gq-dependent phospholipase C causes activation ofgenes containing AP1 elements; thus, activated Gq-associated receptorswill evidence an increase in the expression of such genes, wherebyinverse agonists thereto will evidence a decrease in such expression,and agonists will evidence an increase in such expression. Commerciallyavailable assays for such detection are available.

3. GPCR Fusion Protein

The use of an endogenous, constitutively activated GPCR or anon-endogenous, constitutively activated GPCR, for use in screening ofcandidate compounds for the direct identification of inverse agonists oragonists provides an interesting screening challenge in that, bydefinition, the receptor is active even in the absence of an endogenousligand bound thereto. Thus, in order to differentiate between, e.g., thenon-endogenous receptor in the presence of a candidate compound and thenon-endogenous receptor in the absence of that compound, with an aim ofsuch a differentiation to allow for an understanding as to whether suchcompound may be an inverse agonist or agonist or have no affect on sucha receptor, in some embodiments it is preferred that an approach beutilized that can enhance such differentiation. In some embodiments, apreferred approach is the use of a GPCR Fusion Protein.

Generally, once it is determined that a non-endogenous GPCR has beenconstitutively activated using the assay techniques set forth above (aswell as others known to the art-skilled), it is possible to determinethe predominant G protein that couples with the endogenous GPCR.Coupling of the G protein to the GPCR provides a signaling pathway thatcan be assessed. In some embodiments it is preferred that screening takeplace using a mammalian expression system, such a system will beexpected to have endogenous G protein therein. Thus, by definition, insuch a system, the non-endogenous, constitutively activated GPCR willcontinuously signal. In some embodiments it is preferred that thissignal be enhanced such that in the presence of, e.g., an inverseagonist to the receptor, it is more likely that it will be able to morereadily differentiate, particularly in the context of screening, betweenthe receptor when it is contacted with the inverse agonist.

The GPCR Fusion Protein is intended to enhance the efficacy of G proteincoupling with the non-endogenous GPCR. The GPCR Fusion Protein ispreferred for screening with either an endogenous, constitutively activeGPCR or a non-endogenous, constitutively activated GPCR because such anapproach increases the signal that is generated in such screeningtechniques. This is important in facilitating a significant “signal tonoise” ratio; such a significant ratio is preferred for the screening ofcandidate compounds as disclosed herein.

The construction of a construct useful for expression of a GPCR FusionProtein is within the purview of those having ordinary skill in the art.Commercially available expression vectors and systems offer a variety ofapproaches that can fit the particular needs of an investigator.Important criteria in the construction of such a GPCR Fusion Proteinconstruct include but are not limited to, that the endogenous GPCRsequence and the G protein sequence both be in-frame (preferably, thesequence for the endogenous GPCR is upstream of the G protein sequence),and that the “stop” codon of the GPCR be deleted or replaced such thatupon expression of the GPCR, the G protein can also be expressed. Otherembodiments include constructs wherein the endogenous GPCR sequence andthe G protein sequence are not in-frame and/or the “stop” codon is notdeleted or replaced. The GPCR can be linked directly to the G protein,or there can be spacer residues between the two (preferably, no morethan about 12, although this number can be readily ascertained by one ofordinary skill in the art). Based upon convenience it is preferred touse a spacer. In some embodiments it is preferred, that the G proteinthat couples to the non-endogenous GPCR will have been identified priorto the creation of the GPCR Fusion Protein construct. Because there areonly a few G proteins that have been identified, it is preferred that aconstruct comprising the sequence of the G protein (i.e., a universal Gprotein construct, see Example 5(a) below) be available for insertion ofan endogenous GPCR sequence therein; this provides for furtherefficiency in the context of large-scale screening of a variety ofdifferent endogenous GPCRs having different sequences.

As noted above, constitutively activated GPCRs that couple to Gi, Gz andGo are expected to inhibit the formation of cAMP making assays basedupon these types of GPCRs challenging (i.e., the cAMP signal decreasesupon activation thus making the direct identification of, e.g., inverseagonists (which would further decrease this signal), challenging. Aswill be disclosed herein, it has been ascertained that for these typesof receptors, it is possible to create a GPCR Fusion Protein that is notbased upon the GPCR's endogenous G protein, in an effort to establish aviable cyclase-based assay. Thus, for example, an endogenous Gi coupledreceptor can be fused to a Gs protein—such a fusion construct, uponexpression, “drives” or “forces” the endogenous GPCR to couple with,e.g., Gs rather than the “natural” Gi protein, such that a cyclase-basedassay can be established. Thus, for Gi, Gz and Go coupled receptors, insome embodiments it is preferred that when a GPCR Fusion Protein is usedand the assay is based upon detection of adenylyl cyclase activity, thatthe fusion construct be established with Gs (or an equivalent G proteinthat stimulates the formation of the enzyme adenylyl cyclase). TABLE DEffect of Effect of cAMP Effect of IP₃ cAMP Effect on IP₃ Productionupon Accumulation Production Accu- Activation of upon Activation uponmulation GPCR (i.e., of GPCR (i.e., contact upon contact constitutiveconstitutive with an with an G activation or activation or InverseInverse protein agonist binding) agonist binding) Agonist Agonist GsIncrease N/A Decrease N/A Gi Decrease N/A Increase N/A Gz Decrease N/AIncrease N/A Go Decrease Increase Increase Decrease Gq N/A Increase N/ADecrease

Equally effective is a G Protein Fusion construct that utilizes a GqProtein fused with a Gs, Gi, Gz or Go Protein. In some embodiments apreferred fusion construct can be accomplished with a Gq Protein whereinthe first six (6) amino acids of the G-protein α-subunit (“Gαq”) isdeleted and the last five (5) amino acids at the C-terminal end of Gαqis replaced with the corresponding amino acids of the Gα of the Gprotein of interest. For example, a fusion construct can have a Gq (6amino acid deletion) fused with a Gi Protein, resulting in a “Gq/GiFusion Construct”. This fusion construct will forces the endogenous Gicoupled receptor to couple to its non-endogenous G protein, Gq, suchthat the second messenger, for example, inositol triphosphate ordiacylgycerol, can be measured in lieu of cAMP production.

4. Co-transfection of a Target Gi Coupled GPCR with a Signal-Enhancer GsCoupled GPCR (cAMP Based Assays)

A Gi coupled receptor is known to inhibit adenylyl cyclase, and,therefore, decreases the level of cAMP production, which can make theassessment of cAMP levels challenging. In some preferred embodiments, aneffective technique in measuring the decrease in production of cAMP asan indication of constitutive activation of a receptor thatpredominantly couples Gi upon activation can be accomplished byco-transfecting a signal enhancer, e.g., a non-endogenous,constitutively activated receptor that predominantly couples with Gsupon activation (e.g., TSHR-A623I, disclosed below), with the Gi linkedGPCR. As is apparent, constitutive activation of a Gs coupled receptorcan be determined based upon an increase in production of cAMP.Constitutive activation of a Gi coupled receptor leads to a decrease inproduction cAMP. Thus, the co-transfection approach is intended toadvantageously exploit these “opposite” affects. For example,co-transfection of a non-endogenous, constitutively activated Gs coupledreceptor (the “signal enhancer”) with the endogenous Gi coupled receptor(the “target receptor”) provides a baseline cAMP signal (i.e., althoughthe Gi coupled receptor will decrease cAMP levels, this “decrease” willbe relative to the substantial increase in cAMP levels established byconstitutively activated Gs coupled signal enhancer). By thenco-transfecting the signal enhancer with a constitutively activatedversion of the target receptor, cAMP would be expected to furtherdecrease (relative to base line) due to the increased functionalactivity of the Gi target (i.e., which decreases cAMP).

Screening of candidate compounds using a cAMP based assay can then beaccomplished, with two ‘changes’ relative to the use of the endogenousreceptor/G-protein fusion: first, relative to the Gi coupled targetreceptor, “opposite” effects will result, i.e., an inverse agonist ofthe Gi coupled target receptor will increase the measured cAMP signal,while an agonist of the Gi coupled target receptor will decrease thissignal; second, as would be apparent, candidate compounds that aredirectly identified using this approach should be assessed independentlyto ensure that these do not target the signal enhancing receptor (thiscan be done prior to or after screening against the co-transfectedreceptors).

G. Medicinal Chemistry

Candidate Compounds

Any molecule known in the art can be tested for its ability to modulate(increase or decrease) the activity of a GPCR of the present invention.For identifying a compound that modulates activity, candidate compoundscan be directly provided to a cell expressing the receptor.

This embodiment of the invention is well suited to screen chemicallibraries for molecules which modulate, e.g., inhibit, antagonize, oragonize, the amount of, or activity of, a receptor. The chemicallibraries can be peptide libraries, peptidomimetic libraries, chemicallysynthesized libraries, recombinant, e.g., phage display libraries, andin vitro translation-based libraries, other non-peptide syntheticorganic libraries, etc. This embodiment of the invention is also wellsuited to screen endogenous candidate compounds comprising biologicalmaterials, including but not limited to plasma and tissue extracts, andto screen libraries of endogenous compounds known to have biologicalactivity.

In some embodiments direct identification of candidate compounds isconducted in conjunction with compounds generated via combinatorialchemistry techniques, whereby thousands of compounds are randomlyprepared for such analysis. The candidate compound may be a member of achemical library. This may comprise any convenient number of individualmembers, for example tens to hundreds to thousand to millions ofsuitable compounds, for example peptides, peptoids and other oligomericcompounds (cyclic or linear), and template-based smaller molecules, forexample benzodiazepines, hydantoins, biaryls, carbocyclic and polycycliccompounds (e.g., naphthalenes, phenothiazines, acridines, steroidsetc.), carbohydrate and amino acid derivatives, dihydropyridines,benzhydryls and heterocycles (e.g., trizines, indoles, thiazolidinesetc.). The numbers quoted and the types of compounds listed areillustrative, but not limiting. Preferred chemical libraries comprisechemical compounds of low molecular weight and potential therapeuticagents.

Exemplary chemical libraries are commercially available from severalsources (ArQule, Tripos/PanLabs, ChemDesign, Pharmacopoeia). In somecases, these chemical libraries are generated using combinatorialstrategies that encode the identity of each member of the library on asubstrate to which the member compound is attached, thus allowing directand immediate identification of a molecule that is an effectivemodulator. Thus, in many combinatorial approaches, the position on aplate of a compound specifies that compound's composition. Also, in oneexample, a single plate position may have from 1-20 chemicals that canbe screened by administration to a well containing the interactions ofinterest. Thus, if modulation is detected, smaller and smaller pools ofinteracting pairs can be assayed for the modulation activity. By suchmethods, many candidate molecules can be screened.

Many diversity libraries suitable for use are known in the art and canbe used to provide compounds to be tested according to the presentinvention. Alternatively, libraries can be constructed using standardmethods. Further, more general, structurally constrained, organicdiversity (e.g., nonpeptide) libraries, can also be used. By way ofexample, a benzodiazepine library (see e.g., Bunin et al., 1994, Proc.Natl. Acad. Sci. USA 91:47084712) may be used.

In another embodiment of the present invention, combinatorial chemistrycan be used to identify modulators of the GPCRs of the presentinvention. Combinatorial chemistry is capable of creating librariescontaining hundreds of thousands of compounds, many of which may bestructurally similar. While high throughput screening programs arecapable of screening these vast libraries for affinity for knowntargets, new approaches have been developed that achieve libraries ofsmaller dimension but which provide maximum chemical diversity. (Seee.g., Matter, 1997, Journal of Medicinal Chemistry 40:1219-1229).

One method of combinatorial chemistry, affinity fingerprinting, haspreviously been used to test a discrete library of small molecules forbinding affinities for a defined panel of proteins. The fingerprintsobtained by the screen are used to predict the affinity of theindividual library members for other proteins or receptors of interest(in the instant invention, the receptors of the present invention). Thefingerprints are compared with fingerprints obtained from othercompounds known to react with the protein of interest to predict whetherthe library compound might similarly react. For example, rather thantesting every ligand in a large library for interaction with a complexor protein component, only those ligands having a fingerprint similar toother compounds known to have that activity could be tested. (See, e.g.,Kauvar et al., 1995, Chemistry and Biology 2:107-118; Kauvar, 1995,Affinity fingerprinting, Pharmaceutical Manufacturing International.8:25-28; and Kauvar, Toxic-Chemical Detection by Pattern Recognition inNew Frontiers in Agrochemical Immunoassay, D. Kurtz. L. Stanker and J.H. Skerritt. Editors, 1995, AOAC: Washington, D.C., 305-312).

In some preferred embodiments, the candidate compound is anhydroxypyrazole derivative. In some preferred embodiments, the candidatecompound is a benzotriazole carboxylic acid or ester derivative.

Candidate Compounds Identified as Modulators

Generally, the results of such screening will be compounds having uniquecore structures; thereafter, these compounds may be subjected toadditional chemical modification around a preferred core structure(s) tofurther enhance the medicinal properties thereof. Such techniques areknown to those in the art and will not be addressed in detail in thispatent document.

H. Pharmaceutical Compositions

The invention provides methods of treatment (and prevention) byadministration to an individual in need of said treatment (orprevention) a therapeutically effect amount of a modulator of theinvention [also see, e.g., PCT Application Number PCT/IB02/01461published as WO 02/066505 on 29 Aug. 2002; the disclosure of each ofwhich is hereby incorporated by reference in its entirety]. In apreferred aspect, the modulator is substantially purified. Theindividual is preferably an animal including, but not limited to animalssuch as cows, pigs, horses, chickens, cats, dogs, rabbits, rats, mice,etc., and is preferably a mammal, and most preferably human.

Modulators of the invention can be administered to non-human animals[see Examples, infra] and/or humans, alone or in pharmaceutical orphysiologically acceptable compositions where they are mixed withsuitable carriers or excipient(s) using techniques well known to thosein the art. Suitable pharmaceutically-acceptable carriers are availableto those in the art; for example, see Remington's PharmaceuticalSciences, 16^(th) Edition, 1980, Mack Publishing Co., (Oslo et al.,eds.).

The pharmaceutical or physiologically acceptable composition is thenprovided at therapeutically effect dose. A therapeutically effectivedose refers to that amount of a modulator sufficient to result inprevention or amelioration of symptoms or physiological status ofmetabolic-related disorders or disorders of lipid metabolism asdetermined illustratively and not by limitation by the methods describedherein.

It is expressly considered that the modulators of the invention may beprovided alone or in combination with other pharmaceutically orphysiologically acceptable compounds. Other compounds for the treatmentof disorders of the invention are currently well known in the art. Oneaspect of the invention encompasses the use according to embodimentsdisclosed herein further comprising one or more agents selected from thegroup consisting of α-glucosidase inhibitor, aldose reductase inhibitor,biguanide, HMG-CoA reductase inhibitor, squalene synthesis inhibitor,fibrate, LDL catabolism enhancer, angiotensin converting enzymeinhibitor, insulin secretion enhancer and thiazolidinedione. In someembodiments the agent is a α-glucosidase inhibitor. In some embodimentsthe α-glucosidase inhibitor is acarbose, voglibose or miglitol. In someembodiments the α-glucosidase inhibitor is voglibose. In someembodiments the agent is an aldose reductase inhibitor. In someembodiments the aldose reductase inhibitor is tolurestat; epalrestat;imirestat; zenarestat; zopolrestat; or sorbinil. In some embodiments theagent is a biguanide. In some embodiments the biguanide is phenformin,metformin or buformin. In some embodiments the biguanide is metformin.In some embodiments the agent is a HMG-CoA reductase inhibitor. In someembodiments the HMG-CoA reductase inhibitor is rosuvastatin,pravastatin, simvastatin, lovastatin, atorvastatin, fluvastatin orcerivastatin. In some embodiments the agent is a fibrate. In someembodiments the fibrate is bezafibrate, beclobrate, binifibrate,ciplofibrate, clinofibrate, clofibrate, clofibric acid, etofibrate,fenofibrate, gemfibrozil, nicofibrate, pirifibrate, ronifibrate,simfibrate, or theofibrate. In some embodiments the agent is anangiotensin converting enzyme inhibitor. In some embodiments theangiotensin converting enzyme inhibitor is captopril, enalapril,alacepril, delapril; ramipril, lisinopril, imidapril, benazepril,ceronapril, cilazapril, enalaprilat, fosinopril, moveltopril,perindopril, quinapril, spirapril, temocapril or trandolapril. In someembodiments the agent is an insulin secretion enhancer. In someembodiments the insulin secretion enhancer is tolbutamide;chlorpropamide; tolazamide; acetohexamide; glycopyramide; glibenclamide;gliclazide; 1-butyl-3-metanilylurea; carbutamide; glibonuride;glipizide; gliquidone; glisoxepid; glybuthiazole; glibuzole;glyhexamide; glymidine; glypinamide; phenbutamide; tolcyclamide,glimepiride, nateglinide, or mitiglinide. In some embodiments the agentis a thiazolidinedione. In some embodiments the thiazolidinedione isrosiglitazone or pioglitazone. In some embodiments the thiazolidinedioneis rosiglitazone. In some embodiments, the agent is human adiponectin ora fragment thereof comprising the globular domain.

In some embodiments the metabolic disorder is selected from the groupconsisting of dyslipidemia, atherosclerosis, coronary heart disease,insulin resistance, obesity, impaired glucose tolerance, atheromatousdisease, hypertension, stroke, Syndrome X, heart disease and type 2diabetes. In some embodiments the metabolic disorder is selected fromthe group consisting of dyslipidemia, atherosclerosis, coronary heartdisease, stroke, insulin resistance and type 2 diabetes. In someembodiments, the disorder of lipid metabolism is selected from the groupconsisting of elevated level of plasma triglycerides, elevated level ofplasma free fatty acids, elevated level of plasma cholesterol, elevatedlevel of LDL-cholesterol, reduced level of HDL-cholesterol, elevatedtotal cholesterol/HDL-cholesterol ratio, and reduced level of plasmadiponectin.

Routes of Administration

Suitable routes of administration include oral, nasal, rectal,transmucosal, or intestinal administration, parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, intrapulmonary (inhaled) or intraocularinjections using methods known in the art. Other particularly preferredroutes of administration are aerosol and depot formulation. Sustainedrelease formulations, particularly depot, of the invented medicamentsare expressly contemplated.

Composition/Formulation

Pharmaceutical or physiologically acceptable compositions andmedicaments for use in accordance with the present invention may beformulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries. Properformulation is dependent upon the route of administration chosen.

Certain of the medicaments described herein will include apharmaceutically or physiologically acceptable carrier and at least onemodulator of the invention. For injection, the agents of the inventionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer such as a phosphate or bicarbonate buffer.For transmucosal administration, penetrants appropriate to the barrierto be permeated are used in the formulation. Such penetrants aregenerally known in the art.

Pharmaceutical or physiologically acceptable preparations that can betaken orally include push-fit capsules made of gelatin, as well as soft,sealed captulse made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with fillers such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forsuch administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs for a nebulizer, with the useof a suitable gaseous propellant, e.g., carbon dioxide. In the case of apressurized aerosol the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin, for ue in an inhaler or insufflator, may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage for, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspension, solutions or emulsions in aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical or physiologically acceptable formulations for parenteraladministration include aqueous solutions of the active compounds inwater-soluble form. Aqueous suspension may contain substances thatincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents that increase the solubility ofthe compounds to allow for the preparation of highly concentratedsolutions.

Alternatively, the active ingredient may be in powder or lyophilizedform for constitution with a suitable vehicle, such as sterilepyrogen-free water, before use.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

In a particular embodiment, the compounds can be delivered via acontrolled release system. In one embodiment, a pump may be used(Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201-240;Buchwald et al., 1980, Surgery 88:507-516; Saudek et al., 1989, N. Engl.J. Med. 321:574-579). In another embodiment, polymeric materials can beused (Medical Applications of Controlled Release, Langer and Wise, eds.,CRC Press, Boca Raton, Fla., 1974; Controlled Drug Bioavailability, DrugProduct Design and Performance, Smolen and Ball, eds., Wiley, New York,1984; Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem.23:61; Levy et al., 1985, Science 228:190-192; During et al., 1989, Ann.Neurol. 25:351-356; Howard et al., 1989, J. Neurosurg. 71:858-863).Other controlled release systems are discussed in the review by Langer(1990, Science 249:1527-1533).

Additionally, the compounds may be delivered using a sustained-releasesystem, such as semipermeable matrices of solid hydrophobic polymerscontaining the therapeutic agent. Various sustained release materialshave been established and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days.

Depending on the chemical nature and the biological stability of thetherapeutic reagent, additional strategies for modulator stabilizationmay be employed.

The pharmaceutical or physiologically acceptable compositions also maycomprise suitable solid or gel phase carriers or excipients. Examples ofsuch carriers or excipients include but are not limited to calciumcarbonate, calcium phosphate, various sugars, starches, cellulosederivatives, gelatin, and polymers such as polyethylene glycols.

Effective Dosage

Pharmaceutical or physiologically acceptable compositions suitable foruse in the present invention include compositions wherein the activeingredients are contained in an effective amount to achieve theirintended purpose. More specifically, a therapeutically effective amountmeans an amount effective to prevent development of or to alleviate theexisting symptoms of the subject being treated. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.

For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. For example, a dose can be formulated in animal modelsto achieve a circulating concentration range that includes orencompasses a concentration point or range shown to antilipolytic in anin vitro system. [See Examples, infra, for in vitro assays and in vivoanimal models.] Such information can be used to more accuratelydetermine useful doses in humans.

A therapeutically effective dose refers to that amount of the compoundthat results in amelioration of symptoms in a patient. Toxicity andtherapeutic efficacy of such compounds can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD₅₀ (the dose lethal to 50% of the testpopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe test population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratiobetween LD₅₀ and ED₅₀. Compounds that exhibit high therapeutic indicesare preferred.

The data obtained from these cell culture assays and animal studies canbe used in formulating a range of dosage for use in humans. The dosageof such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀, with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition. (See, e.g.,Fingl et al., 1975, in “The Pharmacological Basis of Therapeutics”, Ch.1).

Dosage amount and interval may be adjusted individually to provideplasma levels of the active compound which are sufficient to prevent ortreat a disorder of the invention, depending on the particularsituation. Dosages necessary to achieve these effects will depend onindividual characteristics and route of administration.

Dosage intervals can also be determined using the value for the minimumeffective concentration. Compounds should be administered using aregimen that maintains plasma levels above the minimum effectiveconcentration for 10-90% of the time, preferably between 30-99%, andmost preferably between 50-90%. In cases of local administration orselective uptake, the effective local concentration of the drug may notbe related to plasma concentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration, and the judgement of theprescribing physician.

A preferred dosage range for the amount of a modulator of the invention,which can be administered on a daily or regular basis to achieve desiredresults, including but not limited to reduction of the level of plasmatriglycerides, reduction of the level of plasma free fatty acids,elevation of the level of HDL-cholesterol, reduction of the level ofLDL-cholesterol, reduction of the level of plasma cholesterol, reductionof the total cholesterol/HDL-cholesterol ratio, or elevation of thelevel of plasma adiponectin, is 0.1-100 mg/kg body mass. Other preferreddosage range is 0.1-30 mg/kg body mass. Other preferred dosage range is0.1-10 mg/kg body mass. Other preferred dosage range is 0.1-3.0 mg/kgbody mass. Of course, these daily dosages can be delivered oradministered in small amounts periodically during the course of a day.It is noted that these dosage ranges are only preferred ranges and arenot meant to be limiting to the invention.

I. Methods of Treatment

The invention is drawn inter alia to methods of preventing or treatingdisorders of lipid metabolism and metabolic-related disorders comprisingproviding an individual in need of such treatment with a modulator ofthe invention. Preferably the modulator is provided to the individual ina pharmaceutical composition that is preferably taken orally. Preferablythe individual is a mammal, and most preferably a human. In preferredembodiments, the disorder of lipid metabolism is selected from the groupconsisting of elevated level of triglycerides, elevated level of plasmafree fatty acids, elevated level of plasma cholesterol, elevated levelof LDL-cholesterol, reduced level of HDL-cholesterol, elevated totalcholesterol/HDL-cholesterol ratio, and reduced level of plasmaadiponectin. In preferred embodiments, the metabolic-related disorder isselected from the group consisting of dyslipidemia, atherosclerosis,coronary heart disease, stroke, insulin resistance, and type 2 diabetes.Other metabolic-related disorders to be treated by modulators of theinvention include obesity, impaired glucose tolerance, atheromatousdisease, hypertension, Syndrome X, and heart disease. Heart diseaseincludes, but is not limited to, cardiac insufficiency, coronaryinsufficiency, and high blood pressure. Other metabolic-related disorderto be treated by modulators of the invention is hyperlipidemia. In otherembodiments, the invention provides for a method of using a modulator ofthe invention as an inhibitor of the progression from impaired glucosetolerance to insulin resistance.

The invention also features methods of preventing or treating disordersof lipid metabolism or metabolic-related disorders comprising providingan individual in need of such treatment with a modulator identified byassays of the invention. Preferably, the modulator is provided to theindividual in a pharmaceutical composition that is preferably takenorally. Preferably the individual is a mammal, and most preferably ahuman. In preferred embodiments, the disorder of lipid metabolism isselected from the group consisting of elevated level of triglycerides,elevated level of plasma free fatty acids, elevated level of plasmacholesterol, elevated level of LDL-cholesterol, reduced level ofHDL-cholesterol, elevated total cholesterol/HDL-cholesterol ratio, andreduced level of plasma adiponectin. In preferred embodiments, themetabolic-related disorder is selected from the group consisting ofdyslipidemia, atherosclerosis, coronary heart disease, stroke, insulinresistance, and type 2 diabetes. Other metabolic-related disorders to betreated by modulators of the invention include obesity, impaired glucosetolerance, atheromatous disease, hypertension, Syndrome X, and heartdisease. Heart disease includes, but is not limited to, cardiacinsufficiency, coronary insufficiency, and high blood pressure. Othermetabolic-related disorder to be treated by modulators of the inventionis hyperlipidemia. In other embodiments, the invention provides for amethod of using a modulator of the invention as an inhibitor of theprogression from impaired glucose tolerance to insulin resistance.

J. Other Utility

Although a preferred use of the non-endogenous versions of the GPCRsdisclosed herein may be for the direct identification of candidatecompounds as inverse agonists or agonists (preferably for use aspharmaceutical agents), other uses of these versions of GPCRs exist. Forexample, in vitro and in vivo systems incorporating GPCRs can beutilized to further elucidate and understand the roles these receptorsplay in the human condition, both normal and diseased, as well asunderstanding the role of constitutive activation as it applies tounderstanding the signaling cascade. In some embodiments it is preferredthat the endogenous receptors be “orphan receptors”, i.e., theendogenous ligand for the receptor has not been identified. In someembodiments, therefore, the modified, non-endogenous GPCRs can be usedto understand the role of endogenous receptors in the human body beforethe endogenous ligand has been identified. Such receptors can be used tofurther elucidate known receptors and the pathways through which theytransduce a signal. The present methods may also be useful in developingtreatment regimens for diseases and disorders associated with thetissues in which the receptors are localized. Examples of such diseasesand disorders and tissues in which the receptors are localized are setforth supra and infra.

Agents that modulate (i.e., increase, decrease, or block) nicotinic acidreceptor functionality may be identified by contacting a candidatecompound with a nicotinic acid receptor and determining the effect ofthe candidate compound on nicotinic acid receptor functionality. Theselectivity of a compound that modulates the functionality of thenicotinic acid receptor can be evaluated by comparing its effects on thenicotinic acid receptor to its effects on other receptors. Followingidentification of compounds that modulate nicotinic acid receptorfunctionality, such candidate compounds may be further tested in otherassays including, but not limited to, in vivo models, in order toconfirm or quantitate their activity. Modulators of nicotinic acidreceptor functionality will be therapeutically useful in treatment ofdiseases and physiological conditions in which normal or aberrantnicotinic acid receptor functionality is involved.

Agents that modulate (i.e., increase, decrease, or block) antilipolyticreceptor functionality may be identified by contacting a candidatecompound with an antilipolytic receptor and determining the effect ofthe candidate compound on antilipolytic receptor functionality. Theselectivity of a compound that modulates the functionality of anantilipolytic receptor can be evaluated by comparing its effects on theantilipolytic receptor to its effects on other receptors. Followingidentification of compounds that modulate antilipolytic receptorfunctionality, such candidate compounds may be further tested in otherassays including, but not limited to, in vivo models, in order toconfirm or quantitate their activity. Modulators of antilipolyticreceptor functionality will be therapeutically useful in treatment ofdiseases and physiological conditions in which normal or aberrantantilipolytic receptor functionality is involved.

Other uses of the disclosed receptors and methods will become apparentto those in the art based upon, inter alia, a review of this patentdocument.

EXAMPLES

The following examples are presented for purposes of elucidation, andnot limitation, of the present invention. While specific nucleic acidand amino acid sequences are disclosed herein, those of ordinary skillin the art are credited with the ability to make minor modifications tothese sequences while achieving the same or substantially similarresults reported below. The traditional approach to application orunderstanding of sequence cassettes from one sequence to another (e.g.from rat receptor to human receptor or from human receptor A to humanreceptor B) is generally predicated upon sequence alignment techniqueswhereby the sequences are aligned in an effort to determine areas ofcommonality. The mutational approach disclosed herein does not rely uponthis approach but is instead based upon an algorithmic approach and apositional distance from a conserved proline residue located within theTM6 region of human GPCRs. Once this approach is secured, those in theart are credited with the ability to make minor modifications thereto toachieve substantially the same results (i.e., constitutive activation)disclosed herein. Such modified approaches are considered within thepurview of this disclosure.

The following Examples are provided for illustrative purposes and not asa means of limitation. One of ordinary skill in the art would be able todesign equivalent assays and methods based on the disclosure herein, allof which form part of the present invention.

Although a variety of expression vectors are available to those in theart, for purposes of utilization for both the endogenous andnon-endogenous human, mouse and rat GPCRs, it is most preferred that thevector utilized be pCMV. This vector was deposited with the AmericanType Culture Collection (ATCC) on Oct. 13, 1998 (10801 University Blvd.,Manassas, Va. 20110-2209 USA) under the provisions of the BudapestTreaty for the International Recognition of the Deposit ofMicroorganisms for the Purpose of Patent Procedure. The DNA was testedby the ATCC and determined to be viable. The ATCC has assigned thefollowing deposit number to pCMV: ATCC #203351. In some alternativeembodiments as relates to said human, mouse and rat GPCRs, it ispreferred that the vector utilized be an adenoviral expression vector.

Recombinant DNA techniques relating to the subject matter of the presentinvention and well known to those of ordinary skill in the art can befound, e.g, in Maniatis T et al., Molecular Cloning: A Laboratory Manual(1989) Cold Spring Harbor Laboratory; U.S. Pat. No. 6,399,373; and PCTApplication Number PCT/IB02/01461 published as WO 02/066505 on 29 Aug.2002; the disclosure of each of which is hereby incorporated byreference in its entirety.

Example 1

A. Endogenous Human GPCRs

Identification of Human GPCRs

The disclosed endogenous human GPCRs were identified based upon a reviewof the GenBank™ database information. While searching the database, thefollowing cDNA clones were identified as evidenced below (Table E).TABLE E Disclosed Human Accession Complete Open Reading Nucleic AcidAmino Acid Orphan Number DNA Sequence Frame SEQ.ID. SEQ.ID. GPCRsIdentified (Base Pairs) (Base Pairs) NO. NO. hRUP8 AL121755 147,566 bp1,152 bp 1 2 hRUP9 AC0113375 143,181 bp 1,260 bp 3 4 hRUP10 AC008745 94,194 bp 1,014 bp 5 6 hRUP11 AC013396 155,086 bp 1,272 bp 7 8 hRUP12AP000808 177,764 bp   966 bp 9 10 hRUP13 AC011780 167,819 bp 1,356 bp 1112 hRUP14 AL137118 168,297 bp 1,041 bp 13 14 hRUP15 AL016468 138,828 bp1,527 bp 15 16 hRUP16 AL136106 208,042 bp 1,068 bp 17 18 hRUP17 AC023078161,735 bp   969 bp 19 20 hRUP18 AC008547 117,304 bp 1,305 bp 21 22hRUP19 AC026331 145,183 bp 1,041 bp 23 24 hRUP20 AL161458 163,511 bp1,011 bp 25 26 hRUP21 AC026756 156,534 bp 1,014 bp 27 28 hRUP22 AC027026151,811 bp   993 bp 29 30 hRUP23 AC007104 200,000 bp 1,092 bp 31 32hRUP24 AL355388 190,538 bp 1,125 bp 33 34 hRUP25 AC026331 145,183 bp1,092 bp 35 36 hRUP26 AC023040 178,508 bp 1,044 bp 37 38 hRUP27 AC027643158,700 bp 1,020 bp 39 40 hRUP38 AC026331 145,183 bp 1,164 bp 134 135

1. Full Length Cloning

a. hRUP8 (Seq. Id. Nos. 1 & 2)

The disclosed human HRUP8 was identified based upon the use of ESTdatabase (dbEST) information. While searching the dbEST, a cDNA clonewith accession number AL121755 was identified to encode a novel GPCR.The following PCR primers were used for RT-PCR with human testisMarathon-Ready cDNA (Clontech) as templates:5′-CTTGCAGACATCACCATGGCAGCC-3′ (SEQ.ID.NO.:41; sense) and5′-GTGATGCTCTGAGTACTGGACTGG-3′. (SEQ.ID.NO.:42; antisense)

PCR was performed using Advantage cDNA polymerase (Clontech;manufacturing instructions will be followed) in 50 ul reaction by thefollowing cycles: 94° C. for 30 sec; 94° C. for 10 sec; 65° C. for 20sec, 72° C. for 1.5 min, and 72° C. for 7 min. Cycles 2 through 4 wererepeated 35 times.

A 1.2 kb PCR fragment was isolated and cloned into the pCRII-TOPO vector(Invitrogen) and sequenced using the ABI Big Dye Terminator kit (P.E.Biosystem). See, SEQ.ID.NO.:1. The putative amino acid sequence forhRUP8 is set forth in SEQ.ID.NO.:2.

b. hRUP9 (Seq. Id. Nos. 3 & 4)

The disclosed human hRUP9 was identified based upon the use of GeneBankdatabase information. While searching the database, a cDNA clone withAccession Number AC01375 was identified as a human genomic sequence fromchromosome 5. The full length hRUP9 was cloned by PCR using primers:5′-GAAGCTGTGAAGAGTGATGC-3′, (SEQ.ID.NO.:43; sense)5′-GTCAGCAATATTGATAAGCAGCAG-3′ (SEQ.ID.NO.:44; antisense)and human genomic DNA (Promega) as a template. Taq Plus Precisionpolymerase (Stratagene) was used for the amplification in a 100 μlreaction with 5% DMSO by the following cycle with step 2 to step 4repeated 35 times: 94° C. for 1 minute; 94° C. for 30 seconds; 56° C.for 30 seconds; 72° C. for 2 minutes; 72° C. for 5 minutes.

A 1.3 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector(Invitrogen) from 1% agarose gel and completely sequenced using the ABIBig Dye Terminator kit (P.E. Biosystems). See, SEQ.ID.NO.:3. Theputative amino acid sequence for hRUP8 is set forth in SEQ.ID.NO.:4. Thesequence of hRUP9 clones isolated from human genomic DNA matched withthe sequence obtained from data base.

c. hRUP10 (Seq. Id. Nos. 5 & 6)

The disclosed human hRUP10 was identified based upon the use of GenBankdatabase information. While searching the database, a cDNA clone withaccession number AC008754 was identified as a human genomic sequencefrom chromosome 19. The full length hRUP10 was cloned by RT-PCR usingprimers: 5′-CCATGGGGAACGATTCTGTCAGCTACG-3′ (SEQ.ID.NO.:45; sense) and5′-GCTATGCCTGAAGCCAGTCTTGTG-3′ (SEQ.ID.NO.:46; antisense)and human leukocyte Marathon-Ready cDNA (Clontech) as a template.Advantage cDNA polymerase (Clontech) was used for the amplification in a50 μl reaction by the following cycle with step 2 to step 4 repeated 35times: 94° C. for 30 seconds; 94° C. for 10 seconds; 62° C. for 20seconds; 72° C. for 1.5 minutes; 72° C. for 7 minutes. A 1.0 Kb PCRfragment was isolated and cloned into the pCRII-TOPO vector (Invitrogen)and completely sequenced using the ABI Big Dye Terminator kit (P.E.Biosystems). The nucleic acid sequence of the novel human receptorhRUP10 is set forth in SEQ.ID.NO.:5 and the putative amino acid sequencethereof is set forth in SEQ.ID.NO.:6.

d. hRUP11 (Seq. Id. Nos. 7 & 8)

The disclosed human hRUP11 was identified based upon the use of GenBankdatabase information. While searching the database, a cDNA clone withaccession number AC013396 was identified as a human genomic sequencefrom chromosome 2. The full length hRUP11 was cloned by PCR usingprimers: 5′-CCAGGATGTTGTGTCACCGTGGTGGC-3′, (SEQ.ID.NO.:47; sense)5′-CACAGCGCTGCAGCCCTGCAGCTGGC-3′ (SEQ.ID.NO.:48; antisense)and human genomic DNA (Clontech) as a template. TaqPlus Precision DNApolymerase (Stratagene) was used for the amplification in a 50 μlreaction by the following cycle with step 2 to step 4 repeated 35 times:94° C. for 3 minutes; 94° C. for 20 seconds; 67° C. for 20 seconds; 72°C. for 1.5 minutes; 72° C. for 7 minutes. A 1.3 Kb PCR fragment wasisolated and cloned into the pCRII-TOPO vector (Invitrogen) andcompletely sequenced using the ABI Big Dye Terminator kit (P.E.Biosystems). The nucleic acid sequence of the novel human receptorhRUP11 is set forth in SEQ.ID.NO.:7 and the putative amino acid sequencethereof is set forth in SEQ.ID.NO.:8.

e. hRUP12 (Seq. Id. Nos. 9 & 10)

The disclosed human HRUP12 was identified based upon the use of GenBankdatabase. While searching the database, a cDNA clone with accessionnumber AP000808 was identified to encode a new GPCR, having significanthomology with rat RTA and human mas1 oncogene GPCRs. The full lengthhRUP12 was cloned by PCR using primers: 5′-CTTCCTCTCGTAGGGATGAACCAGAC-3′(SEQ.ID.NO.:49; sense) 5′-CTCGCACAGGTGGGAAGCACCTGTGG-3′ (SEQ.ID.NO.:50;antisense)and human genomic DNA (Clontech) as template. TaqPlus Precision DNApolymerase (Stratagene) was used for the amplification by the followingcycle with step 2 to step 4 repeated 35 times: 94° C. for 3 min; 94° C.for 20 sec; 65° C. for 20 sec; 72° C. for 2 min and 72° C. for 7 min. A1.0 kb PCR fragment was isolated and cloned into the pCRII-TOPO vector(Invitrogen) and sequenced using the ABI Big Dye Terminator kit (P.E.Biosystems) (see, SEQ.ID.NO.:9 for nucleic acid sequence andSEQ.ID.NO.:10 for deduced amino acid sequence).

f. hRUP13 (Seq. Id. Nos. 11 & 12)

The disclosed human HRUP13 was identified based upon the use of GenBankdatabase. While searching the database, a cDNA clone with accessionnumber AC011780 was identified to encode a new GPCR, having significanthomology with GPCR fish GPRX-ORYLA. The full length hRUP13 was cloned byPCR using primers: 5′-GCCTGTGACAGGAGGTACCCTGG-3′ (SEQ.ID.NO.:51; sense)5′-CATATCCCTCCGAGTGTCCAGCGGC-3′ (SEQ.ID.NO.:52; antisense)and human genomic DNA (Clontech) as template. TaqPlus Precision DNApolymerase (Stratagene) was used for the amplification by the followingcycle with step 2 to step 4 repeated 35 times: 94° C. for 3 min; 94° C.for 20 sec; 65° C. for 20 sec; 72° C. for 2 min and 72° C. for 7 min. A1.35 kb PCR fragment was isolated and cloned into the pCRII-TOPO vector(Invitrogen) and completely sequenced using the ABI Big Dye Terminatorkit (P.E. Biosystems) (see, SEQ.ID.NO.:11 for nucleic acid sequence andSEQ.ID.NO.:12 for deduced amino acid sequence).

g. hRUP14 (Seq. Id. Nos. 13 & 14)

The disclosed human hRUP14 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AL137118 was identified as a human genomicsequence from chromosome 13. The full length hRUP14 was cloned by PCRusing primers: 5′-GCATGGAGAGAAAATTTATGTCCTTGCAACC-3′ (SEQ.ID.NO.:53;sense) 5′-CAAGAACAGGTCTCATCTAAGAGCTCC-3′ (SEQ.ID.NO.:54; antisense)and human genomic DNA (Promega) as a template. Taq Plus Precisionpolymerase (Stratagene) and 5% DMSO were used for the amplification bythe following cycle with step 2 and step 3 repeated 35 times: 94° C. for3 minute; 94° C. for 20 seconds; 58° C. for 2 minutes; 72° C. for 10minutes.

A 1.1 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector(Invitrogen) and completely sequenced using the ABI Big Dye Terminatorkit (P.E. Biosystems) (see, SEQ.ID.NO.:13 for nucleic acid sequence andSEQ.ID.NO.:14 for deduced amino acid sequence). The sequence of hRUP14clones isolated from human genomic DNA matched with the sequenceobtained from database.

h. hRUP15 (Seq. Id. Nos. 15 & 16)

The disclosed human hRUP15 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AC016468 was identified as a human genomicsequence. The full length hRUP15 was cloned by PCR using primers:5′-GCTGTTGCCATGACGTCCACCTGCAC-3′ (SEQ.ID.NO.:55; sense)5′-GGACAGTTCAAGGTTTGCCTTAGAAC-3′ (SEQ.ID.NO.:56; antisense)and human genomic DNA (Promega) as a template. Taq Plus Precisionpolymerase (Stratagene) was used for the amplification by the followingcycle with step 2 to 4 repeated 35 times: 94° C. for 3 minute; 94° C.for 20 seconds; 65° C. for 20 seconds; 72° C. for 2 minutes and 72° C.for 7 minutes.

A 1.5 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector(Invitrogen) and completely sequenced using the ABI Big Dye Terminatorkit (P.E. Biosystems). See, SEQ.ID.NO.:15 for nucleic acid sequence andSEQ.ID.NO.:16 for deduced amino acid sequence. The sequence of hRUP15clones isolated from human genomic DNA matched with the sequenceobtained from database.

i. hRUP16 (Seq. Id. Nos. 17 & 18)

The disclosed human hRUP16 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AL136106 was identified as a human genomicsequence from chromosome 13. The full length hRUP16 was cloned by PCRusing primers: 5′-CTTTCGATACTGCTCCTATGCTC-3′, (SEQ.ID.NO.:57; sense,5′ of initiation codon) 5′-GTAGTCCACTGAAAGTCCAGTGATCC-3′ (SEQ.ID.NO.:58;antisense, 3′ of stop codon)and human skeletal muscle Marathon-Ready cDNA (Clontech) as template.Advantage cDNA polymerase (Clontech) was used for the amplification in a50 ul reaction by the following cycle with step 2 to 4 repeated 35times: 94° C. for 30 seconds; 94° C. for 5 seconds; 69° C. for 15seconds; 72° C. for 1 minute and 72° C. for 5 minutes.

A 1.1 Kb PCR fragment was isolated and cloned into the pCRII-TOPO vector(Invitrogen) and completely sequenced using the T7 Sequenase kit(Amersham). See, SEQ.ID.NO.:17 for nucleic acid sequence andSEQ.ID.NO.:18 for deduced amino acid sequence. The sequence of HRUP16clones matched with four unordered segments of AL136106, indicating thatthe hRUP16 cDNA is composed of 4 exons.

j. hRUP17 (Seq. Id. Nos. 19 & 20)

The disclosed human hRUP17 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AC023078 was identified as a human genomicsequence from chromosome 11. The full length hRUP17 was cloned by PCRusing primers: 5′-TTTCTGAGCATGGATCCAACCATCTC-3′ (SEQ.ID.NO.:59; sense,containing initiation codon) 5′-CTGTCTGACAGGGCAGAGGCTCTTC-3′(SEQ.ID.NO.:60; antisense, 3′of stop codon)and human genomic DNA (Promega) as template. Advantage cDNA polymerasemix (Clontech) was used for the amplification in a 100 μl reaction with5% DMSO by the following cycle with step 2 to 4 repeated 30 times: 94°C. for 1 min; 94° C. for 15 sec; 67° C. for 20 sec; 72° C. for 1 min and30 sec; and 72° C. for 5 min.

A 970 bp PCR fragment was isolated from a 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and completely sequenced using theABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:19 fornucleic acid sequence and SEQ.ID.NO.:20 for deduced amino acid sequence.

k. hRUP18 (Seq. Id. Nos. 21 & 22)

The disclosed human hRUP18 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AC008547 was identified as a human genomicsequence from chromosome 5. The full length hRUP18 was cloned by PCRusing primers: 5′-GGAACTCGTATAGACCCAGCGTCGCTCC-3′, (SEQ.ID.NO.:61;sense, 5′ of the initiation codon) 5′-GGAGGTTGCGCCTTAGCGACAGATGACC-3′(SEQ.ID.NO.:62; antisense, 3′ of stop codon)and human genomic DNA (Promega) as template. TaqPlus precision DNApolymerase (Stratagene) was used for the amplification in a 100 μlreaction with 5% DMSO by the following cycle with step 2 to 4 repeated35 times: 95° C. for 5 min; 95° C. for 30 sec; 65° C. for 30 sec; 72° C.for 2 min; and 72° C. for 5 min.

A 1.3 kb PCR fragment was isolated from a 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and completely sequenced using theABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:21 fornucleic acid sequence and SEQ.ID.NO.:22 for deduced amino acid sequence.

1. hRUP19 (Seq. Id. Nos. 23 & 24)

The disclosed human hRUP19 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AC026331 was identified as a human genomicsequence from chromosome 12. The full length hRUP19 was cloned by PCRusing primers:

5′-CTGCACCCGGACACTTGCTCTG-3′ (SEQ.ID.NO.:63; sense, 5′ of initiationcodon),

5′-GTCTGCTTGTTCAGTGCCACTCAAC-3′ (SEQ.ID.NO.:64; antisense, containingthe stop codon) and human genomic DNA (Promega) as template. TaqPlusPrecision DNA polymerase (Stratagene) was used for the amplificationwith 5% DMSO by the following cycle with step 2 to 4 repeated 35 times:94° C. for 1 min; 94° C. for 15 sec; 70° C. for 20 sec; 72° C. for 1 minand 30 sec; and 72° C. for 5 min.

A 1.1 kp PCR fragment was isolated from a 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and completely sequenced using theABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:23 fornucleic acid sequence and SEQ.ID.NO.:24 for deduced amino acid sequence.

m. hRUP20 (Seq. Id. Nos. 25 & 26)

The disclosed human hRUP20 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AL161458 was identified as a human genomicsequence from chromosome 1. The full length hRUP20 was cloned by PCRusing primers:

5′-TATCTGCAATTCTATTCTAGCTCCTG-3′ (SEQ.ID.NO.:65; sense, 5′ of initiationcodon),

5′-TGTCCCTAATAAAGTCACATGAATGC-3′ (SEQ.ID.NO.:66; antisense, 3′ of stopcodon) and human genomic DNA (Promega) as template. Advantage cDNApolymerase mix (Clontech) was used for the amplification with 5% DMSO bythe following cycle with step 2 to 4 repeated 35 times: 94° C. for 1min; 94° C. for 15 sec; 60° C. for 20 sec; 72° C. for 1 min and 30 sec;and 72° C. for 5 min.

A 1.0 kp PCR fragment was isolated from a 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and completely sequenced using theABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:25 fornucleic acid sequence and SEQ.ID.NO.:26 for deduced amino acid sequence.

n. hRUP21 (Seq. Id. Nos. 27 & 28)

The disclosed human hRUP21 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AC026756 was identified as a human genomicsequence from chromosome 13. The full length hRUP21 was cloned by PCRusing primers: 5′-GGAGACAACCATGAATGAGCCAC-3′ (SEQ.ID.NO.:67; sense)5′-TATTTCAAGGGTTGTTTGAGTAAC-3′ (SEQ.ID.NO.:68; antisense)and human genomic DNA (Promega) as template. Taq Plus Precisionpolymerase (Stratagene) was used for the amplification in a 100 μlreaction with 5% DMSO by the following cycle with step 2 to 4 repeated30 times: 94° C. for 1 min; 94° C. for 15 sec; 55° C. for 20 sec; 72° C.for 1 min and 30 sec; and 72° C. for 5 min.

A 1,014 bp PCR fragment was isolated from a 1% agarose gel and clonedinto the pCRII-TOPO vector (Invitrogen) and completely sequenced usingthe ABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:27 fornucleic acid sequence and SEQ.ID.NO.:28 for deduced amino acid sequence.

o. hRUP22 (Seq. Id. Nos. 29 & 30)

The disclosed human hRUP22 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AC027026 was identified as a human genomicsequence from chromosome 11. The full length hRUP22 was cloned by PCRusing primers:

5′-GGCACCAGTGGAGGTTTTCTGAGCATG-3′ (SEQ.ID.NO.:69; sense, containinginitiation codon)

5′-CTGATGGAAGTAGAGGCTGTCCATCTC-3′ (SEQ.ID.NO.:70; antisense, 3′ of stopcodon)

and human genomic DNA (Promega) as template. TaqPlus Precision DNApolymerase (Stratagene) was used for the amplification in a 100 μlreaction with 5% DMSO by the following cycle with step 2 to 4 repeated30 times: 94° C., 1 minutes 94° C., 15 seconds 55° C., 20 seconds 72°C., 1.5 minute 72° C., 5 minutes.

A 970 bp PCR fragment was isolated from a 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and completely sequenced using theABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:29 fornucleic acid sequence and SEQ.ID.NO.:30 for deduced amino acid sequence.

p. hRUP23 (Seq. Id. Nos. 31 & 32)

The disclosed human hRUP23 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AC007104 was identified as a human genomicsequence from chromosome 4. The full length hRUP23 was cloned by PCRusing primers:

5′-CCTGGCGAGCCGCTAGCGCCATG-3′ (SEQ.ID.NO.:71; sense, ATG as theinitiation codon), 5′-ATGAGCCCTGCCAGGCCCTCAGT-3′ (SEQ.ID.NO.:72;antisense, TCA as the stop codon) and human placenta Marathon-Ready cDNA(Clontech) as template. Advantage cDNA polymerase (Clontech) was usedfor the amplification in a 50 ul reaction by the following cycle withstep 2 to 4 repeated 35 times: 95° C. for 30 sec; 95° C. for 15 sec; 66°C. for 20 sec; 72° C. for 1 min and 20 sec; and 72° C. for 5 min.

A 1.0 kb PCR fragment was isolated and cloned into the pCRII-TOPO vector(Invitrogen) and completely sequenced using the ABI Big Dye TerminatorKit (P.E. Biosystems). See, SEQ.ID.NO.:31 for nucleic acid sequence andSEQ.ID.NO.:32 for deduced amino acid sequence.

q. hRUP24 (Seq. Id. Nos. 33 & 34)

The disclosed human hRUP24 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AL355388 was identified as a human genomicsequence from chromosome 1. The full length hRUP24 was cloned by PCRusing primers:

5′-CTGCGATGCCCACACTCAATACTTCTG-3′ (SEQ.ID.NO.:73; sense, 5′ ofinitiation codon),

5′-AAGGATCCTACACTTGGTGGATCTCAG-3′ (SEQ.ID.NO.:74; antisense, 3′ of stopcodon) and human genomic DNA (Promega) as template. Advantage cDNApolymerase mix (Clontech) was used for the amplification with 5% DMSO bythe following cycle with step 2 to 4 repeated 35 times: 94° C. for 1minute; 94° C. for 15 seconds; 56° C. for 20 seconds 72° C. for 1 minute30 seconds and 72° C. for 5 minutes.

A 1.2 kb PCR fragment was isolated from a 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and completely sequenced using theABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:33 fornucleic acid sequence and SEQ.ID.NO.:34 for deduced amino acid sequence.

r. hRUP25 (Seq. Id. Nos. 35 & 36)

The disclosed human hRUP25 was identified based upon the use of theGenBank database information. While searching the database, a cDNA clonewith Accession Number AC026331 was identified as a human genomicsequence from chromosome 12. The full length hRUP25 was cloned by PCRusing primers:

5′-GCTGGAGCATTCACTAGGCGAG-3′ (SEQ.ID.NO.:75; sense, 5′ of initiationcodon),

5′-AGATCCTGGTTCTTGGTGACAATG-3′ (SEQ.ID.NO.:76; antisense, 3′ of stopcodon) and human genomic DNA (Promega) as template. Advantage cDNApolymerase mix (Clontech) was used for the amplification with 5% DMSO bythe following cycle with step 2 to 4 repeated 35 times: 94° C. for 1minute; 94° C. for 15 seconds; 56° C. for 20 seconds 72° C. for 1 minute30 seconds and 72° C. for 5 minutes.

A 1.2 kb PCR fragment was isolated from a 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and completely sequenced using theABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:35 fornucleic acid sequence and SEQ.ID.NO.:36 for deduced amino acid sequence.

s. hRUP26 (Seq. Id. Nos. 37 & 38)

The disclosed human hRUP26 was identified based upon the use of GenBankdatabase information. While searching the database, a cDNA clone withAccession Number AC023040 was identified as a human genomic sequencefrom chromosome 2. The full length hRUP26 was cloned by RT-PCR usinghRUP26 specific primers:

5′-AGCCATCCCTGCCAGGAAGCATGG-3′ (SEQ.ID.NO.:77; sense, containinginitiation codon)

5′-CCAGACTGTGGACTCAAGAACTCTAGG-3′ (SEQ.ID.NO.:78; antisense, containingstop codon)

and human pancreas Marathon—Ready cDNA (Clontech) as template. AdvantagecDNA polymerase mix (Clontech) was used for the amplification in a 10011reaction with 5% DMSO by the following cycle with step 2 to 4 repeated35 times: 94° C. for 5 minute; 95° C. for 30 seconds; 65° C. for 30seconds 72° C. for 2 minute and 72° C. for 5 minutes.

A 1.1 kb PCR fragment was isolated from 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and completely sequenced using theABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:37 fornucleic acid sequence and SEQ.ID.NO.:38 for deduced amino acid sequence.

t. hRUP27 (Seq. Id. Nos. 39 & 40)

The disclosed human hRUP27 was identified based upon the use of GenBankdatabase information. While searching the database, a cDNA clone withAccession Number AC027643 was identified as a human genomic sequencefrom chromosome 12. The full length hRUP27 was cloned by PCR usinghRUP27 specific primers:

5′-AGTCCACGAACAATGAATCCATITCATG-3′ (SEQ.ID.NO.:79; sense, containinginitiation codon),

5′-ATCATGTCTAGACTCATGGTGATCC-3′ (SEQ.ID.NO.:80; antisense, 3′ of stopcodon)

and the human adult brain Marathon-Ready cDNA (Clontech) as template.Advantage cDNA polymerase mix (Clontech) was used for the amplificationin a 5011 reaction with 5% DMSO by the following cycle with step 2 to 4repeated 35 times: 94° C. for 1 minute; 94° C. for 10 seconds; 58° C.for 20 seconds 72° C. for 1 minute 30 seconds and 72° C. for 5 minutes.

A 1.1 kb PCR fragment was isolated from 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and completely sequenced using theABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:35 fornucleic acid sequence and SEQ.ID.NO.:36 for deduced amino acid sequence.The sequence of hRUP27 cDNA clone isolated from human brain wasdetermined to match with five unordered segments of AC027643, indicatingthat the hRUP27 cDNA is composed of 5 exons.

a. hRUP38 (Seq. Id. Nos. 134 & 135)

The disclosed human hRUP38 was identified based upon the use of GenBankdatabase information. While searching the database, a cDNA clone wasidentified as a human genomic sequence from chromosome 12. The fulllength hRUP38 was cloned by PCR using hRUP38 specific primers:

5′-GCACTCATGAATCGGCACCA-3′ (SEQ.ID.NO.:148; sense, containing initiationcodon),

5′-CAGTGACATTACTCGATGCA-3′ (SEQ.ID.NO.:149; antisense, 3′ of stop codon)

and human genomic DNA (Promega) as template. Advantage cDNA polymerasemix (Clontech) was used for the amplification in a 50 μl reaction with5% DMSO by the following cycle with step 2 to 4 repeated 35 times: 94°C. for 1 minute; 94° C. for 10 seconds; 60° C. for 20 seconds 72° C. for1 minute 30 seconds and 72° C. for 5 minutes.

A 1.2 kb PCR fragment was isolated from 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and completely sequenced using theABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:134 fornucleic acid sequence and SEQ.ID.NO.:135 for deduced amino acidsequence. The sequence of hRUP38 DNA clone isolated from human genomicDNA was determined to match with one genomic sequence on chromosome 12and is without introns.

B. Endogenous Mouse and Rat GPCRS

1. Identification of Mouse and Rat GPCRs

The mouse and rat orthologs of hRUP25 and the mouse ortholog of hRUP19have been identified and are disclosed below as determined from genomicsequence. The rat ortholog of hRUP19 has also been identified (PCTApplication Number PCT/US02/04397, published as WO 02/83736 on Oct. 24,2002; said disclosure is hereby incorporated by reference in itsentirety) and is provided below.

Evidence to date suggests that there is no mouse or rat ortholog ofhRUP38. As the hRUP25 polynucleotide sequence is about 95% identical tohRUP38 polynucleotide sequence, as hRUP25 and hRUP38 are found on thesame arm of chromosome 12, and as an hRUP38 ortholog is absent fromrodents, one may hypothesize without wishing to be bound by theory thathRUP38 was the product of gene duplication. This event must havehappened subsequent to the divergence of human from rodents. PossiblyhRUP38 represents a novel antilipolytic regulatory pathway for whichthere is no counterpart in rodent.

Evidence to date suggests that there may also be no mouse or ratortholog of hRUP11. TABLE F Disclosed Mouse (m) Accession Complete OpenReading Nucleic Acid Amino Acid and Rat (r) Number DNA Sequence FrameSEQ.ID. SEQ.ID. GPCRs Identified (Base Pairs) (Base Pairs) NO. NO. mRU25AJ300199 — 1,083 bp 136 137 rRUP25 None — 1,086 bp 138 139 mRUP19XM_144529 — 1,032 bp 150 151 rRUP19 None — 1,056 bp 156 157

2. Full Length Cloning

a. mRUP25 (Seq. Id. Nos. 136 & 137)

In order to clone the open reading frame encoding the mouse RUP25receptor we applied a PCR based cloning strategy. Primers were designedand synthesized based on the start and stop codon sequence of the mousePUMA-g sequence, published on Genbank, and used on mouse genomic DNA(Promega). The PCR primers were as follows:5′-ATGAGCAAGTCAGACCATTTTCTAGTGATA-3′ (SEQ.ID.NO.:140; sense)5′-TTATCTGGCTTCCACATCTCGTTAA-3′ (SEQ.ID.NO.:141; antisense)Advantage cDNA polymerase mix (Clontech) was used for the amplificationwith 5% DMSO by the following cycle with step 2 to 4 repeated 35 times:94° C. for 1 minute; 94° C. for 15 seconds; 56° C. for 20 seconds 72° C.for 1 minute 30 seconds and 72° C. for 5 minutes.

A 1.2 kb PCR fragment was isolated from a 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and completely sequenced using theABI Big Dye Terminator Kit (P.E. Biosystems). See, SEQ.ID.NO.:35 fornucleic acid sequence and SEQ.ID.NO.:36 for deduced amino acid sequence.

b. rRUP25 (Seq. Id. Nos. 138 & 139)

The rat RUP25 receptor was cloned in an analogous fashion, however thiswas done assuming the sequence would be similar to the mouse sequencebecause there is no previously published rat sequence. Again, we applieda PCR based cloning strategy. Primers were designed and synthesizedbased on the start and stop codon sequence of the mouse PUMA-g sequence,published on Genbank, and used on rat genomic DNA (Promega). The PCRprimers were as follows: 5′-ATGAGCAAGTCAGACCATTTTCTAGTGATA-3′(SEQ.ID.NO.:142; sense) 5′-TTATCTGGCTTCCACATCTCGTTAA-3′ (SEQ.ID.NO.:143;antisense)Cloned Pfu polymerase was used for the amplification by the followingcycle with step 2 to 4 repeated 35 times: 94° C. for 1 minute; 94° C.for 30 sec; 55° C. for 1 min; 72° C. for 2 min; and a final extension at72° C. for 10 minutes.

A 1.2 kb PCR fragment was isolated from a 1% agarose gel and cloned intothe pCRII-TOPO vector (Invitrogen) and 12 clones were completelysequenced using the ABI Big Dye Terminator Kit (P.E. Biosystems).

Example 2 Preparation of Non-Endogenous, Constitutively Activated GPCRS

Those skilled in the art are credited with the ability to selecttechniques for mutation of a nucleic acid sequence. Presented below areapproaches utilized to create non-endogenous versions of several of thehuman GPCRs disclosed above. The mutations disclosed below are basedupon an algorithmic approach whereby the 16^(th) amino acid (located inthe IC3 region of the GPCR) from a conserved proline (or an endogenous,conservative substitution therefor) residue (located in the TM6 regionof the GPCR, near the TM6/IC3 interface) is mutated, preferably to analanine, histamine, arginine or lysine amino acid residue, mostpreferably to a lysine amino acid residue.

1. Transformer Site-Directed™ Mutagenesis

Preparation of non-endogenous human GPCRs may be accomplished on humanGPCRs using, inter alia, Transformer Site-Directed™ Mutagenesis Kit(Clontech) according to the manufacturer instructions. Two mutagenesisprimers are utilized, most preferably a lysine mutagenesisoligonucleotide that creates the lysine mutation, and a selection markeroligonucleotide. For convenience, the codon mutation to be incorporatedinto the human GPCR is also noted, in standard form (Table G): TABLE GReceptor Identifier Codon Mutation hRUP8 V274K hRUP9 T249K hRUP10 R232KhRUP11 M294K hRUP12 F220K hRUP16 A238K hRUP17 Y215K hRUP18 L294K hRUP19T219K hRUP20 K248A K248H K248R hRUP21 R240K hRUP22 Y222K hRUP24 A245KhRUP25 I230K hRUP26 V285K hRUP27 T248K

2. QuikChange™ Site-Directed™ Mutagenesis

Preparation of non-endogenous human GPCRs can also be accomplished byusing QuikChange™ Site-Directed™ Mutagenesis Kit (Stratagene, accordingto manufacturer's instructions). Endogenous GPCR is preferably used as atemplate and two mutagenesis primers utilized, as well as, mostpreferably, a lysine mutagenesis oligonucleotide and a selection markeroligonucleotide (included in kit). For convenience, the codon mutationincorporated into the novel human GPCR and the respectiveoligonucleotides are noted, in standard form (Table H): TABLE H Cycle5′-3′ Conditions orientation Min (′), (sense), 5′-3′ Sec (″)(SEQ.ID.NO.) orientation Cycles 2-4 Receptor Codon mutation (antisense)repeated Identifier Mutation underlined (SEQ.ID.NO.) 16 times hRUP13A268K GGGGAGGGAAAGCA CCAGGAGAACCACC 98° for 2′ AAGGTGGTCCTCCTTTTGCTTTCCCTCC 98° for 30″ GG CC 56° C. for 30″ (81) (82) 72° for11′ 40″ 72° for 5′ hRUP14 L246K CAGGAAGGCAAAGA GATGATGATGGTGG 98° for 2′CCACCATCATCATC TCTTTGCCTTCCTG 98° for 30″ (85) (86) 55° C. for 30″72° for 11′ 40″ 72° for 5′ hRUP15 A398K CCAGTGCAAAGCTA GAAGATCACTTTCT98° for 2′ AGAAAGTGATCTTC TAGCTTTGCACTGG 98° for 30″ (89) (90) 55° C.for 30″ 72° for 11′ 40″ 72° for 5′ hRUP23 W275K GCCGCCACCGCGCCGCCAATCTTCCTCT 98° for 2′ AAGAGGAAGATTGG TGGCGCGGTGGCGG 98° for 30″ C C56° C. for 30″ (93) (94) 72° for 11′ 40″ 72° for 5′

The non-endogenous human GPCRs were then sequenced and the derived andverified nucleic acid and amino acid sequences are listed in theaccompanying “Sequence Listing” appendix to this patent document, assummarized in Table I below: TABLE I Non Endogenous Nucleic Acid AminoAcid Human GPCR Sequence Listing Sequence Listing hRUP13 SEQ.ID.NO.:83SEQ.ID.NO.:84 hRUP14 SEQ.ID.NO.:87 SEQ.ID.NO.:88 hRUP15 SEQ.ID.NO.:91SEQ.ID.NO.:92 hRUP23 SEQ.ID.NO.:95 SEQ.ID.NO.:96

Example 3 Receptor Expression

Although a variety of cells are available to the art for the expressionof proteins, it is most preferred that mammalian cells be utilized. Theprimary reason for this is predicated upon practicalities, i.e.,utilization of, e.g., yeast cells for the expression of a GPCR, whilepossible, introduces into the protocol a non-mammalian cell which maynot (indeed, in the case of yeast, does not) include thereceptor-coupling, genetic-mechanism and secretary pathways that haveevolved for mammalian systems—thus, results obtained in non-mammaliancells, while of potential use, are not as preferred as that obtainedfrom mammalian cells. Of the mammalian cells, COS-7, 293 and 293T cellsare particularly preferred, although the specific mammalian cellutilized can be predicated upon the particular needs of the artisan.

a. Transient Transfection

On day one, 6×10⁶/10 cm dish of 293 cells well were plated out. On daytwo, two reaction tubes were prepared (the proportions to follow foreach tube are per plate): tube A was prepared by mixing 4 μg DNA (e.g.,pCMV vector; pCMV vector with receptor cDNA, etc.) in 0.5 ml serum freeDMEM (Gibco BRL); tube B was prepared by mixing 2411 lipofectamine(Gibco BRL) in 0.5 ml serum free DMEM. Tubes A and B were admixed byinversions (several times), followed by incubation at room temperaturefor 30-45 min. The admixture is referred to as the “transfectionmixture”. Plated 293 cells were washed with 1×PBS, followed by additionof 5 ml serum free DMEM. 1 ml of the transfection mixture was added tothe cells, followed by incubation for 4 hrs at 37° C./5% CO₂. Thetransfection mixture was removed by aspiration, followed by the additionof 10 ml of DMEM/10% Fetal Bovine Serum. Cells were incubated at 37°C./5% CO₂. After 48 hr incubation, cells were harvested and utilized foranalysis.

b. Stable Cell Lines: Gs Fusion Protein

Approximately 12×10⁶ 293 cells are plated on a 15 cm tissue cultureplate. Grown in DME High Glucose Medium containing ten percent fetalbovine serum and one percent sodium pyruvate, L-glutamine, andantibiotics. Twenty-four hours following plating of 293 cells (or to˜80% confluency), the cells are transfected using 12 μg of DNA. The 12μg of DNA is combined with 60 μl of lipofectamine and 2 mL of DME HighGlucose Medium without serum. The medium is aspirated from the platesand the cells are washed once with medium without serum. The DNA,lipofectamine, and medium mixture are added to the plate along with 10mL of medium without serum. Following incubation at 37 degrees Celsiusfor four to five hours, the medium is aspirated and 25 ml of mediumcontaining serum is added. Twenty-four hours following transfection, themedium is aspirated again, and fresh medium with serum is added.Forty-eight hours following transfection, the medium is aspirated andmedium with serum is added containing geneticin (G418 drug) at a finalconcentration of 500 μg/mL. The transfected cells now undergo selectionfor positively transfected cells containing the G418 resistant gene. Themedium is replaced every four to five days as selection occurs. Duringselection, cells are grown to create stable pools, or split for stableclonal selection.

Example 4 Assays for Determination of Constitutive Activity ofNon-Endogenous GPCRs

A variety of approaches are available for assessment of constitutiveactivity of the non-endogenous human GPCRs. The following areillustrative; those of ordinary skill in the art are credited with theability to determine those techniques that are preferentially beneficialfor the needs of the artisan.

1. Membrane Binding Assays: [³⁵S]GTPγS Assay

When a G protein-coupled receptor is in its active state, either as aresult of ligand binding or constitutive activation, the receptorcouples to a G protein and stimulates the release of GDP and subsequentbinding of GTP to the G protein. The alpha subunit of the Gprotein-receptor complex acts as a GTPase and slowly hydrolyzes the GTPto GDP, at which point the receptor normally is deactivated.Constitutively activated receptors continue to exchange GDP for GTP. Thenon-hydrolyzable GTP analog, [³⁵S]GTPγS, can be utilized to demonstrateenhanced binding of [³⁵S]GTPγS to membranes expressing constitutivelyactivated receptors. The advantage of using [³⁵S]GTPγS binding tomeasure constitutive activation is that:

(a) it is generically applicable to all G protein-coupled receptors; (b)it is proximal at the membrane surface making it less likely to pick-upmolecules which affect the intracellular cascade.

The assay utilizes the ability of G protein coupled receptors tostimulate [³⁵S]GTPγS binding to membranes expressing the relevantreceptors. The assay can, therefore, be used in the directidentification method to screen candidate compounds to known, orphan andconstitutively activated G protein-coupled receptors. The assay isgeneric and has application to drug discovery at all G protein-coupledreceptors.

The [³⁵]GTPγS assay was incubated in 20 mM HEPES and between 1 and about20 mM MgCl₂ (this amount can be adjusted for optimization of results,although 20 mM is preferred) pH 7.4, binding buffer with between about0.3 and about 1.2 nM [³⁵S]GTPγS (this amount can be adjusted foroptimization of results, although 1.2 is preferred) and 12.5 to 75 μgmembrane protein (e.g, 293 cells expressing the Gs Fusion Protein; thisamount can be adjusted for optimization) and 10 μM GDP (this amount canbe changed for optimization) for 1 hour. Wheatgerm agglutinin beads (25μl; Amersham) were then added and the mixture incubated for another 30minutes at room temperature. The tubes were then centrifuged at 1500×gfor 5 minutes at room temperature and then counted in a scintillationcounter.

2. Adenylyl Cyclase

A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No.SMP004A) designed for cell-based assays can be modified for use withcrude plasma membranes. The Flash Plate wells can contain a scintillantcoating which also contains a specific antibody recognizing cAMP. ThecAMP generated in the wells can be quantitated by a direct competitionfor binding of radioactive cAMP tracer to the cAMP antibody. Thefollowing serves as a brief protocol for the measurement of changes incAMP levels in whole cells that express the receptors.

Transfected cells were harvested approximately twenty four hours aftertransient transfection. Media is carefully aspirated off and discarded.10 ml of PBS is gently added to each dish of cells followed by carefulaspiration. 1 ml of Sigma cell dissociation buffer and 3 ml of PBS areadded to each plate. Cells were pipetted off the plate and the cellsuspension was collected into a 50 ml conical centrifuge tube. Cellswere then centrifuged at room temperature at 1,100 rpm for 5 min. Thecell pellet was carefully re-suspended into an appropriate volume of PBS(about 3 ml/plate). The cells were then counted using a hemocytometerand additional PBS was added to give the appropriate number of cells(with a final volume of about 50 μl/well).

cAMP standards and Detection Buffer (comprising 1 μCi of tracer [¹²⁵IcAMP (50 μl] to 11 ml Detection Buffer) was prepared and maintained inaccordance with the manufacturer's instructions. Assay Buffer wasprepared fresh for screening and contained 50 μl of Stimulation Buffer,3 ul of test compound (12 μM final assay concentration) and 50 μl cells,Assay Buffer was stored on ice until utilized. The assay was initiatedby addition of 50 μl of cAMP standards to appropriate wells followed byaddition of 50 ul of PBSA to wells H-11 and H12. 50 μl of StimulationBuffer was added to all wells. DMSO (or selected candidate compounds)was added to appropriate wells using a pin tool capable of dispensing 3μl of compound solution, with a final assay concentration of 12 μM testcompound and 100 μl total assay volume. The cells were then added to thewells and incubated for 60 min at room temperature. 100 μl of DetectionMix containing tracer cAMP was then added to the wells. Plates were thenincubated additional 2 hours followed by counting in a Wallac MicroBetascintillation counter. Values of cAMP/well were then extrapolated from astandard cAMP curve which was contained within each assay plate.

3. Cell-Based cAMP for Gi Coupled Target GPCRs

TSHR is a Gs coupled GPCR that causes the accumulation of cAMP uponactivation. TSHR will be constitutively activated by mutating amino acidresidue 623 (i.e., changing an alanine residue to an isoleucineresidue). A Gi coupled receptor is expected to inhibit adenylyl cyclase,and, therefore, decrease the level of cAMP production, which can makeassessment of cAMP levels challenging. An effective technique formeasuring the decrease in production of cAMP as an indication ofconstitutive activation of a Gi coupled receptor can be accomplished byco-transfecting, most preferably, non-endogenous, constitutivelyactivated TSHR (TSHR-A623ID (or an endogenous, constitutively active Gscoupled receptor) as a “signal enhancer” with a Gi linked target GPCR toestablish a baseline level of cAMP. Upon creating a non-endogenousversion of the Gi coupled receptor, this non-endogenous version of thetarget GPCR is then co-transfected with the signal enhancer, and it isthis material that can be used for screening. We will utilize suchapproach to effectively generate a signal when a cAMP assay is used;this approach is preferably used in the direct identification ofcandidate compounds against Gi coupled receptors. It is noted that for aGi coupled GPCR, when this approach is used, an inverse agonist of thetarget GPCR will increase the cAMP signal and an agonist will decreasethe cAMP signal.

On day one, 2×10⁴ 293 cells/well will be plated out. On day two, tworeaction tubes will be prepared (the proportions to follow for each tubeare per plate): tube A will be prepared by mixing 2 μg DNA of eachreceptor transfected into the mammalian cells, for a total of 4 μg DNA(e.g., pCMV vector; pCMV vector with mutated THSR (TSHR-A623ID;TSHR-A623I and GPCR, etc.) in 1.2 ml serum free DMEM (Irvine Scientific,Irvine, Calif.); tube B will be prepared by mixing 120 μl lipofectamine(Gibco BRL) in 1.2 ml serum free DMEM. Tubes A and B will then beadmixed by inversions (several times), followed by incubation at roomtemperature for 30-45 min. The admixture is referred to as the“transfection mixture”. Plated 293 cells will be washed with 1×PBS,followed by addition of 10 ml serum free DMEM. 2.4 ml of thetransfection mixture will then be added to the cells, followed byincubation for 4 hrs at 37° C./5% CO₂. The transfection mixture willthen be removed by aspiration, followed by the addition of 25 ml ofDMEM/10% Fetal Bovine Serum. Cells will then be incubated at 37° C./5%CO₂. After 24 hr incubation, cells will then be harvested and utilizedfor analysis.

A Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat. No.SMP004A) is designed for cell-based assays, however, can be modified foruse with crude plasma membranes depending on the need of the skilledartisan. The Flash Plate wells will contain a scintillant coating whichalso contains a specific antibody recognizing cAMP. The cAMP generatedin the wells can be quantitated by a direct competition for binding ofradioactive cAMP tracer to the cAMP antibody. The following serves as abrief protocol for the measurement of changes in cAMP levels in wholecells that express the receptors.

Transfected cells will be harvested approximately twenty four hoursafter transient transfection. Media will be carefully aspirated off anddiscarded. 10 ml of PBS will be gently added to each dish of cellsfollowed by careful aspiration. 1 ml of Sigma cell dissociation bufferand 3 ml of PBS will be added to each plate. Cells will be pipetted offthe plate and the cell suspension will be collected into a 50 ml conicalcentrifuge tube. Cells will then be centrifuged at room temperature at1,100 rpm for 5 min. The cell pellet will be carefully re-suspended intoan appropriate volume of PBS (about 3 ml/plate). The cells will then becounted using a hemocytometer and additional PBS is added to give theappropriate number of cells (with a final volume of about 50 μl/well).

cAMP standards and Detection Buffer (comprising 1 μCi of tracer [¹²⁵IcAMP (50 μl] to 11 ml Detection Buffer) will be prepared and maintainedin accordance with the manufacturer's instructions. Assay Buffer shouldbe prepared fresh for screening and contained 50 μl of StimulationBuffer, 3 μl of test compound (12 μM final assay concentration) and 50μl cells, Assay Buffer can be stored on ice until utilized. The assaycan be initiated by addition of 50 μl of cAMP standards to appropriatewells followed by addition of 50 μl of PBSA to wells H-11 and H12. Fiftyμl of Stimulation Buffer will be added to all wells. Selected compounds(e.g., TSH) will be added to appropriate wells using a pin tool capableof dispensing 3 μl of compound solution, with a final assayconcentration of 12 μM test compound and 100 μl total assay volume. Thecells will then be added to the wells and incubated for 60 min at roomtemperature. 100 μl of Detection Mix containing tracer cAMP will then beadded to the wells. Plates were then incubated additional 2 hoursfollowed by counting in a Wallac MicroBeta scintillation counter. Valuesof cAMP/well will then be extrapolated from a standard cAMP curve whichis contained within each assay plate.

4. Reporter-Based Assays

a. CRE-LUC Reporter Assay (Gs-Associated Receptors)

293 and 293T cells are plated-out on 96 well plates at a density of2×10⁴ cells per well and were transfected using Lipofectamine Reagent(BRL) the following day according to manufacturer instructions. ADNA/lipid mixture is prepared for each 6-well transfection as follows:260 ng of plasmid DNA in 100 μl of DMEM were gently mixed with 2 μl oflipid in 100 μl of DMEM (the 260 ng of plasmid DNA consisted of 200 ngof a 8xCRE-Luc reporter plasmid, 50 ng of pCMV comprising endogenousreceptor or non-endogenous receptor or pCMV alone, and 10 ng of a GPRSexpression plasmid (GPRS in pcDNA3 (Invitrogen)). The 8XCRE-Luc reporterplasmid was prepared as follows: vector SRIF-β-gal was obtained bycloning the rat somatostatin promoter (−71/+51) at BglV-HindIII site inthe pβgal-Basic Vector (Clontech). Eight (8) copies of cAMP responseelement were obtained by PCR from an adenovirus template AdpCF126CCRE8(see, 7 Human Gene Therapy 1883 (1996)) and cloned into the SRIF-β-galvector at the Kpn-BglV site, resulting in the 8xCRE-β-gal reportervector. The 8xCRE-Luc reporter plasmid was generated by replacing thebeta-galactosidase gene in the 8xCRE-β-gal reporter vector with theluciferase gene obtained from the pGL3-basic vector (Promega) at theHindIII-BamHI site. Following 30 min. incubation at room temperature,the DNA/lipid mixture was diluted with 400 μl of DMEM and 100 μl of thediluted mixture was added to each well. 100 μl of DMEM with 10% FCS wereadded to each well after a 4 hr incubation in a cell culture incubator.The following day the transfected cells were changed with 200 μl/well ofDMEM with 10% FCS. Eight (8) hours later, the wells were changed to 100μl/well of DMEM without phenol red, after one wash with PBS. Luciferaseactivity were measured the next day using the LucLite™ reporter geneassay kit (Packard) following manufacturer instructions and read on a1450 MicroBeta™ scintillation and luminescence counter (Wallac).

b. AP1 Reporter Assay (Gq-Associated Receptors)

A method to detect Gq stimulation depends on the known property ofGq-dependent phospholipase C to cause the activation of genes containingAP1 elements in their promoter. A Pathdetect™ AP-1 cis-Reporting System(Stratagene, Catalogue # 219073) can be utilized following the protocolset forth above with respect to the CREB reporter assay, except that thecomponents of the calcium phosphate precipitate were 410 ng pAP1-Luc, 80ng pCMV-receptor expression plasmid, and 20 ng CMV-SEAP.

C. SRF-Luc Reporter Assay (Gq-Associated Receptors)

One method to detect Gq stimulation depends on the known property ofGq-dependent phospholipase C to cause the activation of genes containingserum response factors in their promoter. A Pathdetect™SRF-Luc-Reporting System (Stratagene) can be utilized to assay for Gqcoupled activity in, e.g., COS7 cells. Cells are transfected with theplasmid components of the system and the indicated expression plasmidencoding endogenous or non-endogenous GPCR using a MammalianTransfection™ Kit (Stratagene, Catalogue #200285) according to themanufacturer's instructions. Briefly, 410 ng SRF-Luc, 80 ngpCMV-receptor expression plasmid and 20 ng CMV-SEAP (secreted alkalinephosphatase expression plasmid; alkaline phosphatase activity ismeasured in the media of transfected cells to control for variations intransfection efficiency between samples) are combined in a calciumphosphate precipitate as per the manufacturer's instructions. Half ofthe precipitate is equally distributed over 3 wells in a 96-well plate,kept on the cells in a serum free media for 24 hours. The last 5 hoursthe cells are incubated with 1 μM Angiotensin, where indicated. Cellsare then lysed and assayed for luciferase activity using a Luclite™ Kit(Packard, Cat. # 6016911) and “Trilux 1450 Microbeta” liquidscintillation and luminescence counter (Wallac) as per themanufacturer's instructions. The data can be analyzed using GraphPadPrism™ 2.0a (GraphPad Software Inc.).

d. Intracellular IP₃ Accumulation Assay (Gq-Associated Receptors)

On day 1, cells comprising the receptors (endogenous and/ornon-endogenous) can be plated onto 24 well plates, usually 1×10⁵cells/well (although his umber can be optimized. On day 2 cells can betransfected by firstly mixing 0.25 μg DNA in 50 μl serum free DMEM/welland 2 μl lipofectamine in 50 μl serum free DMEM/well. The solutions aregently mixed and incubated for 15-30 min at room temperature. Cells arewashed with 0.5 ml PBS and 400 μl of serum free media is mixed with thetransfection media and added to the cells. The cells are then incubatedfor 3-4 hrs at 37° C./5% CO₂ and then the transfection media is removedand replaced with 1 ml/well of regular growth media. On day 3 the cellsare labeled with ³H-myo-inositol. Briefly, the media is removed and thecells are washed with 0.5 ml PBS. Then 0.5 ml inositol-free/serum freemedia (GIBCO BRL) is added/well with 0.25 μCi of ³H-myo-inositol/welland the cells are incubated for 16-18 hrs o/n at 37° C./5% CO₂. On Day 4the cells are washed with 0.5 ml PBS and 0.45 ml of assay medium isadded containing inositol-free/serum free media 10 μM pargyline 10 mMlithium chloride or 0.4 ml of assay medium and 50 μl of 10 x ketanserin(ket) to final concentration of 10 μM. The cells are then incubated for30 min at 37° C. The cells are then washed with 0.5 ml PBS and 200 μl offresh/ice cold stop solution (1M KOH; 18 mM Na-borate; 3.8 mM EDTA) isadded/well. The solution is kept on ice for 5-10 min or until cells werelysed and then neutralized by 200 μl of fresh/ice cold neutralizationsol. (7.5% HCL). The lysate is then transferred into 1.5 ml eppendorftubes and 1 ml of chloroform/methanol (1:2) is added/tube. The solutionis vortexed for 15 sec and the upper phase is applied to a BioradAG1-X8™ anion exchange resin (100-200 mesh). Firstly, the resin iswashed with water at 1:1.25 W/V and 0.9 ml of upper phase is loaded ontothe column. The column is washed with 10 mls of 5 mM myo-inositol and 10ml of 5 mM Na-borate/60 mM Na-formate. The inositol tris phosphates areeluted into scintillation vials containing 10 ml of scintillationcocktail with 2 ml of 0.1 M formic acid/1 M ammonium formate. Thecolumns are regenerated by washing with 10 ml of 0.1 M formic acid/3Mammonium formate and rinsed twice with dd H₂O and stored at 4° C. inwater.

Exemplary results are presented below in Table J: TABLE J Difference (

<) Signal Between Signal Generated: 1. CMV v. Generated: Non- Wild-Assay Endogenous Endogenous type Utilized Signal Version Version 2.Wild- (Figure Generated: (Relative (Relative type Receptor Mutation No.)CMV Light Units) Light Units) v. Mutant hRUP12 N/A IP₃ 317.03 cpm/mg3463.29 cpm/mg — 1. 11 Fold

(FIG. 1) protein protein hRUP13 N/A cAMP 8.06 pmol/cAMP/ 19.10pmol/cAMP/ — 1. 2.4 Fold

(FIG. 2) mg protein mg protein A268K 8XCRE- 3665.43 LPCS 83280.17 LPCS61713.6 LCPS 1. 23 Fold

LUC 2. 26% < (FIG. 3) hRUP14 L246K 8XCRE- 86.07 LCPS 1962.87 LCPS 789.73LCPS 1. 23 Fold

LUC 2. 60% < (FIG. 5) hRUP15 A398K 8XCRE- 86.07 LCPS 18286.77 LCPS17034.83 LCPS 1. 212 Fold

LUC 2. 1% < (FIG. 6) A398K cAMP 15.00 pmol/cAMP/ 164.4 pmol/cAMP/ 117.5pmol/cAMP/ 1. 11 Fold

(FIG. 7) mg protein mg protein mg protein 2. 29% < hRUP17 N/A IP₃ 317.03cpm/mg 741.07 cpm/ — 1. 2.3 Fold

(FIG. 9) protein mg protein hRUP21 N/A IP₃ 730.5 cpm/mg 1421.9 cpm/mg— 1. 2 Fold

(FIG. 10) protein protein hRUP23 W275K 8XCRE- 311.73 pmol/cAMP/ 13756.00pmol/cAMP/ 9756.87 pmol/cAMP/ 1. 44 Fold

LUC mg protein protein mg protein 2. 30% < (FIG. 11)N/A = not applied

Exemplary results of GTPγS assay for detecting constitutive activation,as disclosed in Example 4(1) above, was accomplished utilizingG_(s):Fusion Protein Constructs on human hRUP13 and hRUP15. Table Kbelow lists the signals generated from this assay and the difference insignals as indicated: TABLE K Difference Between: 1. CMV v. FusionSignal Protein Signal Signal Generated: 2. CMV + GDP Generated:Generated: Fusion vs. Signal Fusion CMV + 10 μM Protein + 10 μM Fusion +GDP Generated: Protein GDP GDP 3. Fusion vs. Receptor: CMV (cpm (cpm(cpm Fusion + GDP Gs Fusion Assay (cpm bound bound bound bound (cpmbound Protein Utilized GTP) GTP) GTP) GTP) GTP) hRUP13- GTPγS 32494.049351.30 11148.30 28834.67 1. 1.5 Fold

Gs (FIG. 2. 2.6 Fold

4) 3. 42% < hRUP15- GTPγS 30131.67 32493.67 7697.00 14157.33 1. 1.1 Fold

Gs (FIG. 2. 1.8 Fold

8) 3. 56% <

Example 5 Fusion Protein Preparation

a. GPCR:Gs Fusion Constuct

The design of the constitutively activated GPCR-G protein fusionconstruct was accomplished as follows: both the 5′ and 3′ ends of therat G protein Gsα (long form; Itoh, H. et al., 83 PNAS 3776 (1986)) wereengineered to include a HindIII (5′-AAGCTT-3′) sequence thereon.Following confirmation of the correct sequence (including the flankingHindIII sequences), the entire sequence was shuttled into pcDNA3.1(−)(Invitrogen, cat. no. V795-20) by subcloning using the HindIIIrestriction site of that vector. The correct orientation for the G_(s)αsequence was determined after subcloning into pcDNA3.1(−). The modifiedpcDNA3.1(−) containing the rat G_(s)α gene at HindIII sequence was thenverified; this vector was now available as a “universal” G_(s)α (proteinvector. The pcDNA3.1 (−) vector contains a variety of well-knownrestriction sites upstream of the HindIII site, thus beneficiallyproviding the ability to insert, upstream of the Gs protein, the codingsequence of an endogenous, constitutively active GPCR. This sameapproach can be utilized to create other “universal” G protein vectors,and, of course, other commercially available or proprietary vectorsknown to the artisan can be utilized—the important criteria is that thesequence for the GPCR be upstream and in-frame with that of the Gprotein.

hRUP13 couples via Gs. For the following exemplary GPCR Fusion Proteins,fusion to Gsα was accomplished.

A hRUP13Gsα Fusion Protein construct was made as follows: primers weredesigned as follows: 5′-gatc[TGTAGAAT]GGAGTCCTCACCCATCCCCCAG-3′ (SEQID.NO.:97; sense) 5′-gatc[GATATC]CGTGACTCCAGCCGGGGTGAGGCGGC-3′.(SEQ.ID.NO.:98; antisense)

Nucleotides in lower caps are included as spacers in the restrictionsites (designated in brackets) between the G protein and hRUP13. Thesense and anti-sense primers included the restriction sites for XbaI andEcoRV, respectively, such that spacers (attributed to the restrictionsites) exist between the G protein and hRUP15.

PCR was then utilized to secure the respective receptor sequences forfusion within the Gsα universal vector disclosed above, using thefollowing protocol for each: 100 ng cDNA for hRUP15 was added toseparate tubes containing 2 μl of each primer (sense and anti-sense), 3μL of 10 mM dNTPs, 10 μL of 10XTaqPlus™ Precision buffer, 1 μL ofTaqPlus™ Precision polymerase (Stratagene: #600211), and 80 μL of water.Reaction temperatures and cycle times for hRUP15 were as follows withcycle steps 2 through 4 were repeated 35 times: 94° C. for 1 min; 94° C.for 30 seconds; 62° C. for 20 sec; 72° C. 1 min 40 sec; and 72° C. 5min. PCR product for was run on a 1% agarose gel and then purified (datanot shown). The purified product was digested with XbaI and EcoRV andthe desired inserts purified and ligated into the Gs universal vector atthe respective restriction site. The positive clones was isolatedfollowing transformation and determined by restriction enzyme digest;expression using 293 cells was accomplished following the protocol setforth infra. Each positive clone for hRUP15-Gs Fusion Protein wassequenced to verify correctness. (See, SEQ.ID.NO.:99 for nucleic acidsequence and SEQ.ID.NO.:100 for amino acid sequence).

hRUP15 couples via Gs. For the following exemplary GPCR Fusion Proteins,fusion to Gsα was accomplished.

A hRUP15-Gsα Fusion Protein construct was made as follows: primers weredesigned as follows: 5′-TCTAGAATGACGTCCACCTGCACCAACAGC-3′(SEQ.ID.NO.:101; sense) 5′-gatatcGCAGGAAAAGTAGCAGAATCGTAGGAAG-3′.(SEQ.ID.NO.:102; antisense)

Nucleotides in lower caps are included as spacers in the restrictionsites between the G protein and hRUP15. The sense and anti-sense primersincluded the restriction sites for EcoRV and Xba1, respectively, suchthat spacers (attributed to the restriction sites) exists between the Gprotein and hRUP15.

PCR was then utilized to secure the respective receptor sequences forfusion within the Gsα universal vector disclosed above, using thefollowing protocol for each: 100 ng cDNA for hRUP15 was added toseparate tubes containing 2 μl of each primer (sense and anti-sense), 3μL of 10 mM dNTPs, 10 μL of 10XTaqPlus™ Precision buffer, 1 uL ofTaqPlus™ Precision polymerase (Stratagene: #600211), and 80 μL of water.Reaction temperatures and cycle times for hRUP15 were as follows withcycle steps 2 through 4 were repeated 35 times: 94° C. for 1 min; 94° C.for 30 seconds; 62° C. for 20 sec; 72° C. for 1 min 40 sec; and 72° C.for 5 min. The PCR product for was run on a 1% agarose gel and thenpurified (data not shown). The purified product was digested. Thepurified product was digested with EcoRV and Xba1 and the desiredinserts purified and ligated into the Gs universal vector at therespective restriction site. The positive clones were isolated followingtransformation and determined by restriction enzyme digest; expressionusing 293 cells was accomplished following the protocol set forth infra.Each positive clone for hRUP15-Gs Fusion Protein was sequenced to verifycorrectness. (See, SEQ.ID.NO.:103 for nucleic acid sequence andSEQ.ID.NO.:104 for amino acid sequence).

b. Gq(6 Amino Acid Deletion)/Gi Fusion Construct

The design of a Gq (del)/Gi fusion construct can be accomplished asfollows: the N-terminal six (6) amino acids (amino acids 2 through 7,having the sequence of TLESIM (SEQ.ID.NO.:129) G_(α)q-subunit will bedeleted and the C-terminal five (5) amino acids, having the sequenceEYNLV (SEQ.ID.NO.:130) will be replaced with the corresponding aminoacids of the G_(α)i Protein, having the sequence DCGLF (SEQ.ID.NO.:131).This fusion construct will be obtained by PCR using the followingprimers: (SEQ.ID.NO.:132) 5′-gatcaagcttcCATGGCGTGCTGCCTGAGCGAGGAG-3′ and(SEQ.ID.NO.:133) 5′-gatcggatccTTAGAACAGGCCGCAGTCCTTCAGGTTCAGCTGCAGGATGGTG-3′and Plasmid 63313 which contains the mouse G_(α)q-wild type version witha hemagglutinin tag as template. Nucleotides in lower caps are includedas spacers.

TaqPlus Precision DNA polymerase (Stratagene) will be utilized for theamplification by the following cycles, with steps 2 through 4 repeated35 times: 95° C. for 2 min; 95° C. for 20 sec; 56° C. for 20 sec; 72° C.for 2 min; and 72° C. for 7 min. The PCR product will be cloned into apCRII-TOPO vector (Invitrogen) and sequenced using the ABI Big DyeTerminator kit (P.E. Biosystems). Inserts from a TOPO clone containingthe sequence of the fusion construct will be shuttled into theexpression vector pcDNA3.1(+) at the HindIII/BamHI site by a 2 stepcloning process.

Example 6 Tissue Distribution of the Disclosed Human GPCRS

A. RT-PCR

RT-PCR was applied to confirm the expression and to determine the tissuedistribution of several novel human GPCRs. Oligonucleotides utilizedwere GPCR-specific and the human multiple tissue cDNA panels (MTC,Clontech) as templates. Taq DNA polymerase (Stratagene) were utilizedfor the amplification in a 40 μl reaction according to themanufacturer's instructions. 20 μl of the reaction will be loaded on a1.5% agarose gel to analyze the RT-PCR products. Table L below lists thereceptors, the cycle conditions and the primers utilized.

By way of illustration, RT-PCR results for hRUP25 and hRUP38 are shownin FIG. 13C. RT-PCR results for hRUP19 are shown in FIG. 27, and RT-PCRresults for mRUP19 are shown in FIG. 29. Applicant discloses herein thathRUP25, hRUP38 and hRUP19 are expressed by primary adipocytes and, inthe case of hRUP38 and hRUP19, have limited tissue distribution beyondadipose. That hRUP19 has limited tissue distribution is further apparentby Northern blot analysis (FIG. 28). TABLE L Cycle Conditions Min (′),Sec (″) Cycles 2-4 Receptor repeated 5′ Primer 3′ Primer DNA TissueIdentifier 30 times (SEQ.ID.NO.) (SEQ.ID.NO.) Fragment Expression hRUP1094° for 30″ CATGTATG GCTATGCCT 730 bp Kidney, 94° for 10″ CCAGCGTCGAAGCCAG leukocyte, 62° C. for 20″ CTGCTCC TCTTGTG liver, 72° for 1′(105) (106) placenta 72° for 7′ and spleen *cycles 2-4 repeated 35 timeshRUP11 94° for 2′ GCACCTGC CACAGCGC 630 bp Liver, kidney, 94° for 15″TCCTGAGC TGCAGCCCT pancreas, 67° C. for 15″ ACCTTCTCC GCAGCTGG colon,small 72° for 45″ (107) C intestinal, 72° for 5′ (108) spleen andprostate hRUP12 94° for 2′ CCAGTGAT CAGACACTT 490 bp Brain, colon,94° for 15″ GACTCTGT GGCAGGGA heart, kidney, 66° C. for 15″ CCAGCCTGCGAGGTG leukocyte, 72° for 45″ (109) (110) pancreas, 72° for 5′prostate, small intestinal, spleen, testis, and thymus hRUP13 94° for 1′CTTGTGGTC CATATGCCT 700 bp Placenta and 94° for 15″ TACTGCAG CCGAGTGTClung 68° C. for 20″ CATGTTCCG CAGCGGC 72° for 1′ 45″ (111) (112) 72° for5′ hRUP14 94° for 1′ ATGGATCC CAAGAACAG 700 bp Not yet 94° for 15″TTATCATG GTCTCATCT determined 68° C. for 20″ GCTTCCTC AAGAGCTCC 72° for1′ 45″ (113) (114) 72° for 5′ hRUP16 94° for 30″ CTCTGATG GTAGTCCAC 370bp Fetal brain, 94° for 5″ CCATCTGCT TGAAAGTC fetal kidney 69° C. for15″ GGATTCCTG CAGTGATCC and fetal 72° for 30″ (115) (116) Skeletal72° for 5′ muscle hRUP18 94° for 2′ TGGTGGCG GTTGCGCCT 330 bp Pancreas94° for 15″ ATGGCCAA TAGCGACA 60° C. for 20″ CAGCGCTC GATGACC 72° for 1′(117) (118) 72° for 5′ hRUP19 95° for 4′ GGCCGTGG AACCGGGT 492 bpAdipose, 95° for 1′ CTGATTTCC CGCCTTCTT adipocyte 60.5° C. for 30″TCCTTAT GATCC 72° for 1′ (152) (153) 72° for 7′ *cycles 2-4 repeated 35times hRUP21 94° for 1′ TCAACCTG AAGGAGTA Kidney, lung 94° for 15″TATAGCAG GCAGAATG and testis 56° C. for 20″ CATCCTC GTTAGCC 72° for 40″(119) (120) *cycles 2-3 repeated 30 times hRUP22 94° for 30″ GACACCTGCTGATGGAA Testis, thymus 94° for 15″ TCAGCGGT GTAGAGGCT and spleen69° C. for 20″ CGTGTGTG GTCCATCTC 72° for 40″ (121) (122) *cycles 2-3repeated 30 times hRUP23 94° for 2′ GCGCTGAG CACGGTGA 520 bp Placenta94° for 15″ CGCAGACC CGAAGGGC 60° C. for 20″ AGTGGCTG ACGAGCTC 72° for1′ (123) (124) 72° for 5′ hRUP25 96° for 2′ CTGATGGA GCTGAAGCT 297 bpAdipocyte, 96° for 30″ CAACTATG GCTGCACAA spleen, 55° C. for 1′ TGAGGCGTATTTGCACC leukocyte, 72° for 2′ TGG (145) kidney, lung, 72° for 10′(144) testis hRuP26 94° for 2′ AGCCATCC CCAGGTAGG 470 bp Pancreas94° for 15″ CTGCCAGG TGTGCAGCA 65° C. for 20″ AAGCATGG CAATGGC 72° for1′ (125) (126) 72° for 5′ hRUP27 94° for 30″ CTGTTCAAC ATCATGTCT 890 bpBrain 94° for 10″ AGGGCTGGT AGACTCAT 55° C. for 20″ TGGCAAC GGTGATCC72° for 1′ (127) (128) 72° for 3′ *cycles 2-4 repeated 35 times hRUP3896° for 2′ CTACTATGTG CCCTTCTTGG 852 bp Adipocyte, 96° for 30″ CGGCGTTCAAATGGTTATTT spleen, lung 55° C. for 1′ (146) (147) 72° for 2′ 72° for10′

B. Affymetrix GeneChip® Technology

Amino acid sequences were submitted to Affymetrix for the designing andmanufacturing of microarray containing oligonucleotides to monitor theexpression levels of G protein-coupled receptors (GPCRs) using theirGeneChip® Technology. Also present on the microarray were probes forcharacterized human brain tissues from Harvard Brain Band or obtainedfrom commercially available sources. RNA samples were amplified,labeled, hybridized to the microarray, and data analyzed according tomanufacturer's instructions.

Adipose tissues were monitored for the level of gene expression of eachof the GPCRs represented on the microarray. GPCRs were determined to beexpressed if the expression index was greater than 100 (based upon andaccording to manufacturer's instructions). The data was analyzed and hadindicated that classification of GPCRs with an expression index greaterthan 100 was reasonable because a number of known GPCRs had previouslybeen reported to be expressed in neuronal tissues with an expressionindex greater than 100.

Using the GeneChip, Applicant has discovered hRUP25 and hRUP38 to havehigh levels of expression in adipocytes, consistent with hRUP25 andhRUP38 playing a role in lipolysis (see, Goodman & Gilman's, ThePharmacological Basis of Therapeutics, 9^(th) Edition, page 235 (1996).See FIGS. 13A and 13B. FIG. 13A is a plot representing the expressionlevel of hRUP25 in various tissues. hRUP25 is highly expressed byprimary adipocytes. FIG. 13B is a plot representing the expression levelof hRUP38 in various tissues. hRUP38 is highly expressed by primaryadipocytes.

This patent document discloses the identification of nicotinic acid as aligand and agonist of human, mouse and rat RUP25. See, Examples infra.The patent document further discloses that nicotinic acid is not anagonist of hRUP38 or hRUP19. In the case of hRUP38, this was anunexpected result, as hRUP25 and hRUP38 are about 95% identical (TableB), although it is not a result without precedent [see, e.g., Yan M etal. Science (2000) 290:523-7; the disclosure of which is herebyincorporated by reference in its entirety].

Example 7 Protocol: Direct Identification of Inverse Agonists andAgonists

A. [³⁵S]GTPγS Assay

Although we have utilized endogenous, constitutively active GPCRs forthe direct identification of candidate compounds as, e.g., inverseagonists, for reasons that are not altogether understood, intra-assayvariation can become exacerbated. In some embodiments, a GPCR FusionProtein, as disclosed above, is also utilized with a non-endogenous,constitutively activated GPCR. When such a protein is used, intra-assayvariation appears to be substantially stabilized, whereby an effectivesignal-to-noise ratio is obtained. This has the beneficial result ofallowing for a more robust identification of candidate compounds. Thus,in some embodiments it is preferred that for direct identification, aGPCR Fusion Protein be used and that when utilized, the following assayprotocols be utilized.

1. Membrane Preparation

In some embodiments membranes comprising the constitutively activeorphan GPCR/Fusion Protein of interest and for use in the directidentification of candidate compounds as inverse agonists or agonistsare preferably prepared as follows:

a. Materials

“Membrane Scrape Buffer” is comprised of 20 mM HEPES and 10 mM EDTA, pH7.4; “Membrane Wash Buffer” is comprised of 20 mM HEPES and 0.1 mM EDTA,pH 7.4; “Binding Buffer” is comprised of 20 mM HEPES, 100 mM NaCl, and10 mM MgCl₂, pH 7.4

b. Procedure

All materials will be kept on ice throughout the procedure. Firstly, themedia will be aspirated from a confluent monolayer of cells, followed byrinse with 10 ml cold PBS, followed by aspiration. Thereafter, 5 ml ofMembrane Scrape Buffer will be added to scrape cells; this will befollowed by transfer of cellular extract into 50 ml centrifuge tubes(centrifuged at 20,000 rpm for 17 minutes at 4° C.). Thereafter, thesupernatant will be aspirated and the pellet will be resuspended in 30ml Membrane Wash Buffer followed by centrifuge at 20,000 rpm for 17minutes at 4° C. The supernatant will then be aspirated and the pelletresuspended in Binding Buffer. This will then be homogenized using aBrinkman Polytron™ homogenizer (15-20 second bursts until the allmaterial is in suspension). This is referred to herein as “MembraneProtein”.

2. Bradford Protein Assay

Following the homogenization, protein concentration of the membraneswill be determined using the Bradford Protein Assay (protein can bediluted to about 1.5 mg/ml, aliquoted and frozen (−80° C.) for lateruse; when frozen, protocol for use will be as follows: on the day of theassay, frozen Membrane Protein is thawed at room temperature, followedby vortex and then homogenized with a Polytron at about 12×1,000 rpm forabout 5-10 seconds; it was noted that for multiple preparations, thehomogenizor should be thoroughly cleaned between homogenization ofdifferent preparations).

a. Materials

Binding Buffer (as per above); Bradford Dye Reagent; Bradford ProteinStandard will be utilized, following manufacturer instructions (Biorad,cat. no. 500-0006).

b. Procedure

Duplicate tubes will be prepared, one including the membrane, and one asa control “blank”. Each contained 800 μl Binding Buffer. Thereafter, 10μl of Bradford Protein Standard (1 mg/ml) will be added to each tube,and 10 μg of membrane Protein will then be added to just one tube (notthe blank). Thereafter, 200 μl of Bradford Dye Reagent will be added toeach tube, followed by vortex of each. After five (5) minutes, the tubeswill be re-vortexed and the material therein will be transferred tocuvettes. The cuvettes will then be read using a CECIL 3041spectrophotometer, at wavelength 595.

3. Direct Identification Assay

a. Materials

GDP Buffer consisted of 37.5 ml Binding Buffer and 2 mg GDP (Sigma, cat.no. G-7127), followed by a series of dilutions in Binding Buffer toobtain 0.2 μM GDP (final concentration of GDP in each well was 0.1 μMGDP); each well comprising a candidate compound, has a final volume of200 μl consisting of 100 μl GDP Buffer (final concentration, 0.1 μMGDP), 50 μl Membrane Protein in Binding Buffer, and 50 μl [³⁵S]GTPγS(0.6 nM) in Binding Buffer (2.5 μl [³⁵S]GTPγS per 10 ml Binding Buffer).

b. Procedure

Candidate compounds will be preferably screened using a 96-well plateformat (these can be frozen at −80° C.). Membrane Protein (or membraneswith expression vector excluding the GPCR Fusion Protein, as control),will be homogenized briefly until in suspension. Protein concentrationwill then be determined using the Bradford Protein Assay set forthabove. Membrane Protein (and control) will then be diluted to 0.25 mg/mlin Binding Buffer (final assay concentration, 12.5 μg/well). Thereafter,100 μl GDP Buffer was added to each well of a Wallac Scintistrip™(Wallac). A 5 ul pin-tool will then be used to transfer 5 μl of acandidate compound into such well (i.e., 5 μl in total assay volume of200 μl is a 1:40 ratio such that the final screening concentration ofthe candidate compound is 10 μM). Again, to avoid contamination, aftereach transfer step the pin tool should be rinsed in three reservoirscomprising water (1X), ethanol (1X) and water (2X)—excess liquid shouldbe shaken from the tool after each rinse and dried with paper andkimwipes. Thereafter, 50 μl of Membrane Protein will be added to eachwell (a control well comprising membranes without the GPCR FusionProtein was also utilized), and pre-incubated for 5-10 minutes at roomtemperature. Thereafter, 50 μl of [³⁵S]GTPγS (0.6 nM) in Binding Bufferwill be added to each well, followed by incubation on a shaker for 60minutes at room temperature (again, in this example, plates were coveredwith foil). The assay will then be stopped by spinning of the plates at4000 RPM for 15 minutes at 22° C. The plates will then be aspirated withan 8 channel manifold and sealed with plate covers. The plates will thenbe read on a Wallac 1450 using setting “Prot. #37” (as per manufacturerinstructions).

B. Cyclic AMP Assay

Another assay approach to directly identified candidate compound wasaccomplished by utilizing a cyclase-based assay. In addition to directidentification, this assay approach can be utilized as an independentapproach to provide confirmation of the results from the [³⁵S]GTPγSapproach as set forth above.

A modified Flash Plate™ Adenylyl Cyclase kit (New England Nuclear; Cat.No. SMP004A) was preferably utilized for direct identification ofcandidate compounds as inverse agonists and agonists to constitutivelyactivated orphan GPCRs in accordance with the following protocol.

Transfected cells were harvested approximately three days aftertransfection. Membranes were prepared by homogenization of suspendedcells in buffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl₂.Homogenization was performed on ice using a Brinkman Polytron™ forapproximately 10 seconds. The resulting homogenate is centrifuged at49,000×g for 15 minutes at 4° C. The resulting pellet was thenresuspended in buffer containing 20 mM HEPES, pH 7.4 and 0.1 mM EDTA,homogenized for 10 seconds, followed by centrifugation at 49,000×g for15 minutes at 4° C. The resulting pellet was then stored at −80° C.until utilized. On the day of direct identification screening, themembrane pellet was slowly thawed at room temperature, resuspended inbuffer containing 20 mM HEPES, pH 7.4 and 10 mM MgCl₂, to yield a finalprotein concentration of 0.60 mg/ml (the resuspended membranes areplaced on ice until use).

cAMP standards and Detection Buffer (comprising 2 μCi of tracer [¹²⁵IcAMP (100 μl] to 11 ml Detection Buffer) were prepared and maintained inaccordance with the manufacturer's instructions. Assay Buffer wasprepared fresh for screening and contained 20 mM HEPES, pH 7.4, 10 mMMgCl₂, 20 mM phospocreatine (Sigma), 0.1 units/ml creatine phosphokinase(Sigma), 50 μM GTP (Sigma), and 0.2 mM ATP (Sigma); Assay Buffer wasthen stored on ice until utilized.

Candidate compounds identified as per above (if frozen, thawed at roomtemperature) were added, preferably, to 96-well plate wells (3 μl/well;12 μM final assay concentration), together with 40 μl Membrane Protein(30 μg/well) and 50 μl of Assay Buffer. This admixture was thenincubated for 30 minutes at room temperature, with gentle shaking.

Following the incubation, 100 μl of Detection Buffer was added to eachwell, followed by incubation for 2-24 hours. Plates were then counted ina Wallac MicroBeta™ plate reader using “Prot. #31” (as per manufacturerinstructions).

A representative screening assay plate (96 well format) result ispresented in FIG. 12. Each bar represents the results for a differentcompound in each well, plus hRUP13-Gsα Fusion Protein construct, asprepared in Example 5(a) above. The representative results presented inFIG. 12 also provide standard deviations based upon the mean results ofeach plate (“m”) and the mean plus two arbitrary preference forselection of inverse agonists as “leads” from the primary screeninvolves selection of candidate compounds that that reduce the percentresponse by at least the mean plate response, minus two standarddeviations. Conversely, an arbitrary preference for selection ofagonists as “leads” from the primary screen involves selection ofcandidate compounds that increase the percent response by at least themean plate response, plus the two standard deviations. Based upon theseselection processes, the candidate compounds in the following wells weredirectly identified as putative inverse agonist (Compound A) and agonist(Compound B) to hRUP13 in wells A2 and G9, respectively. See, FIG. 12.It is noted for clarity: these compounds have been directly identifiedwithout any knowledge of the endogenous ligand for this GPCR. Byfocusing on assay techniques that are based upon receptor function, andnot compound binding affinity, we are able to ascertain compounds thatare able to reduce the functional activity of this receptor (Compound A)as well as increase the functional activity of the receptor (CompoundB). Based upon the location of these receptors in, for example, lungtissue (see, for example, hRUP13 and hRUP21 in Example 6),pharmaceutical agents can be developed for potential therapeutictreatment of lung cancer.

Example 8 Melanophore Technology

Melanophores are skin cells found in lower vertebrates. They containpigmented organelles termed melanosomes. Melanophores are able toredistribute these melanosomes along a microtubule network uponG-protein coupled receptor (GPCR) activation. The result of this pigmentmovement is an apparent lightening or darkening of the cells. Inmelanophores, the decreased levels of intracellular cAMP that resultfrom activation of a Gi-coupled receptor cause melanosomes to migrate tothe center of the cell, resulting in a dramatic lightening in color. IfcAMP levels are then raised, following activation of a Gs-coupledreceptor, the melanosomes are re-dispersed and the cells appear darkagain. The increased levels of diacylglycerol that result fromactivation of Gq-coupled receptors can also induce this re-dispersion.In addition, the technology is also suited to the study of certainreceptor tyrosine kinases. The response of the melanophores takes placewithin minutes of receptor activation and results in a simple, robustcolor change. The response can be easily detected using a conventionalabsorbance microplate reader or a modest video imaging system. Unlikeother skin cells, the melanophores derive from the neural crest andappear to express a full complement of signaling proteins. Inparticular, the cells express an extremely wide range of G-proteins andso are able to functionally express almost all GPCRs.

Melanophores can be utilized to identify compounds, including naturalligands, against GPCRs. This method can be conducted by introducing testcells of a pigment cell line capable of dispersing or aggregating theirpigment in response to a specific stimulus and expressing an exogenousclone coding for the GCPR. A stimulant, e.g., melatonin, sets an initialstate of pigment disposition wherein the pigment is aggregated withinthe test cells if activation of the GPCR induces pigment dispersion.However, stimulating the cell with a stimulant to set an initial stateof pigment disposition wherein the pigment is dispersed if activation ofthe GPCR induces pigment aggregation. The test cells are then contactedwith chemical compounds, and it is determined whether the pigmentdisposition in the cells changed from the initial state of pigmentdisposition. Dispersion of pigments cells due to the candidate compound,including but not limited to a ligand, coupling to the GPCR will appeardark on a petri dish, while aggregation of pigments cells will appearlight.

Materials and methods will be followed according to the disclosure ofU.S. Pat. No. 5,462,856 and U.S. Pat. No. 6,051,386. These patentdisclosures are hereby incorporated by reference in their entirety.

Melanophores were transfected by electroporation with plasmids codingfor the GPCRs, for example hRUP25, hRUP38, hRUP11 and hRUP19.Pre-screening of the GPCRs in melanophores was performed in the absenceof nicotinic acid following the protocol below to determine the Gprotein coupling. This pre-screen evidenced that hRUP25 (FIG. 14A),hRUP38 (FIG. 14B) and hRUP19 (FIG. 14C) are strongly Gi-coupled. hRUP11is also strongly Gi-coupled (not shown). Consistent with hRUP19 beingGi-coupled, CART-activated hRUP19 inhibits cAMP production in membranesof transfected 293 cells (FIG. 30).

The cells were plated in 96-well plates (one receptor per plate). 48hours post-transfection, half of the cells on each plate were treatedwith 10 nM melatonin. Melatonin activates an endogenous Gi-coupledreceptor in the melanophores and causes them to aggregate their pigment.The remaining half of the cells were transferred to serum-free medium0.7X L-15 (Gibco). After one hour, the cells in serum-free mediaremained in a pigment-dispersed state while the melatonin-treated cellswere in a pigment-aggregated state. At this point, the cells weretreated with a dose response of nicotinic acid (Sigma). If the platedGPCRs bound to nicotinic acid, the melanophores would be expected toundergo a color change in response to the compound. If the receptor wereeither a Gs or Gq coupled receptor, then the melatonin-aggregatedmelanophores would undergo pigment dispersion. In contrast, if thereceptor was a Gi-coupled receptor, then the pigment-dispersed cellswould be expected to undergo a dose-dependent pigment aggregation.

Melanophores transfected with hRUP25 were treated with nicotinic acid.Upon this treatment, the cells underwent pigment aggregation in adose-dependent manner. hRUP25-expressing cells that were pre-aggregatedwith melatonin did not disperse upon nicotinic acid treatment, which isconsistent with the receptor being a Gi-coupled receptor. See, FIG. 15and infra.

To confirm and extend these results, melanophores were transfected witha range of hRUP25 DNA from 0 to 10% g. As controls, melanophores werealso transfected with 10 μg of α_(2A) Adrenergic receptor (a knownGi-coupled receptor) and salmon sperm DNA (Gibco), as a mocktransfection. On day 3, the cells were again incubated for 1 hour inserum-free L-15 medium (Gibco) and remained in a pigment-dispersedstate. The cells were then treated with a dose response of nicotinicacid. See, FIG. 15A. FIG. 15A depicts the aggregation response ofnicotinic acid at melanophores transfected with various ranges ofhRUP25. At 10 μg of hRUP25, the EC₅₀ for nicotinic acid is about 54 nM.Stated differently, at very low concentrations, nicotinic acid evidencesbinding to hRUP25.

Reference is now made to FIG. 15B. In FIG. 15B, both the mocktransfected and α_(2A) transfected cells did not respond to nicotinicacid. This data evidences that nicotinic acid binds specifically to theGi-coupled receptor hRUP25.

The data show that the greater the amount of hRUP25 plasmid DNAtransfected, the greater the magnitude of the observed aggregationresponse. Collectively these data indicate that hRUP25: 1) is aGi-coupled receptor that 2) binds to nicotinic acid.

As set forth herein, nicotinic acid is a ligand for, and agonist of,human, mouse and rat RUP25. It is further shown that hRUP38, hRUP11,hRUP19, and human, mouse and rat RUP25 are Gi-coupled. Additionally,hRUP38, human and mouse RUP19, hRUP11, and human, mouse, and rat RUP25may be used in methods described herein to identify antagonists,agonists, inverse agonists, partial agonists, allosteric enhancers, andnegative allosteric modulators. As discussed supra, methods of modifyingnicotinic acid receptor activity in adipocytes using a modulator of thereceptor are set forth. Preferably, the modulator is an agonist.

Example 9 Nicotinic Acid Induced-Inositol Phosphates Accumulation in 293Cells Co-Expressing hRUP AND GqΔGi

FIG. 16 illustrates the nicotinic acid induced-inositol phosphates (IPs)accumulation in HEK293 cells co-expressing hRUP25 and the chimericGαq-subunit in which the last five amino acids have been replaced withthe corresponding amino acids of Gαi (GqΔGi). This construct has beenshown to convert the signaling of a Gi-coupled receptor to the Gqpathway (i.e. accumulation of inositol phosphates) in response toreceptor activation. Cells transfected with GqΔGi plus either emptyplasmid or the constitutively activated α_(2A)AR (α_(2A)K) arenon-responsive to nicotinic acid and served as controls for the IPassay. Cells transfected with GqΔGi plus either hRUP19 or hRUP38 arealso unresponsive to nicotinic acid, indicating that nicotinic acid isnot an agonist for either hRUP19 or hRUP38.

Example 10 Saturation Binding of [³H] Nicotinic Acid to Membranes fromCells Expressing Either hRUP25, hRUP38, hRUP190R Vector Alone

FIG. 17 shows the results from saturation binding of [3H] nicotinic acidto membranes from cells expressing either hRUP25, hRUP38, hRUP19 orvector alone [CHO(−)]. Only hRUP25 was found to bind nicotinic acid in aspecific and high-affinity manner.

Example 11 Nicotinic Acid and (−)-Nicotine Induced-Inhibition ofForskolin Stimulated cAMP Accumulation in hRUP25-CHO Cell Stable Line#46

FIG. 18A is a set of immunofluorescent photomicrographs illustrating theexpression of hemaglutinin(HA)-tagged hRUP25 in a stably transfectedline of CHO cells (top; clone #46). No significant labeling is detectedin mock stably-transfected CHO cells (Mock). The lower panels identifythe nuclear (DAPI) staining of cells in the same field.

FIG. 18B illustrates nicotinic acid and (−)-nicotine induced-inhibitionof forskolin stimulated cAMP accumulation in hRUP25-CHO cell stable line#46 (described in preceding paragraph). The EC₅₀ for nicotinic acid is23.6 nM and that for (−)-nicotine is 9.8 μM.

Example 12 hRUP25 AND mRUP25 Inhibit TSHR Induced-Camp AccumulationFollowing Activation by Nicotinic Acid

FIG. 19 indicates that, in response to nicotinic acid, both hRUP25 andthe mouse ortholog mRUP25 can inhibit TSHR stimulated cAMP production(in the presence and absence of TSH).

Example 13 hRUP25 AND mRUP25 Bind to Nicotinic Acid Specifically andwith High Affinity

FIG. 20 shows the saturation binding curves of [³H]nicotinic acid([³H]NA) to membranes prepared from HEK293 cells transiently expressingeither hRUP25 or mRUP25. Note the significant binding of [³H]NA relativeto either that found in membranes derived from mock transfected cells orin the presence of an excess of non-labeled nicotinic acid (200 μM).

Radioligand binding was done as follows. Media was removed from cellsgrown in culture [either stably or transiently transfected with negativecontrol (empty plasmid) or with the individual receptors hRUP25, mRUP25,rRUP25, hRUP38, hRUP11 or hRUP19] and cells were scraped and homogenizedin buffer containing 15 mM HEPES, 5 mM EDTA, 5 mM EGTA, plus proteaseinhibitors (leupeptin, PMSF and pepstatin). Membranes were harvestedfollowing centrifugation at 30,000×g, 4° C. for 30 min. Membranes werethen resuspended and re-homogenized in CHAPS binding buffer (50 mMTris-HCl and 0.02% CHAPS, pH 7.4). Aliquots were taken for proteinanalysis via the Bradford protein assay and normalized such that eachbinding reaction contained the same amount of membrane protein (25-50%g). 50 nM [³H]nicotinic acid was added to each sample and either buffer(for total samples) or a desired amount of non-labeled compound (at thesame volumes and in the same diluent) was added and the reactions wereleft at room temperature gently shaking for 1 hr. Free ligand wasseparated from bound ligand via rapid filtration onto a filter.Appropriate scintilant was added to each sample and counted in anappropriate scintillation counter. Data was analyzed using Excel andPrismGraph. In some cases radioligand binding was performed using ascintillation proximity assay (SPA) in which case the samples did notrequire filtration or the addition of scintilant.

Example 14 The Rank Order of Potency of Compounds on HRUP25 CloselyMatches that of the Pharmacologically Defined Nicotinic Acid Receptor

FIG. 21 is a table comparing the rank order of potency of variouscompounds on hRUP25 and the pharmacologically defined nicotinic acidreceptor. The potencies at hRUP25 derived both by a functional analysismeasuring the inhibition of forskolin induced cAMP production andcompetitive radioligand binding assays, closely match the order ofpotencies of the pharmacologically defined nicotinic acid receptor.

Example 15 Nicotinic Acid and Related Compounds Inhibit IsoproterenolInduced Lipolysis in Rat Epididymal Fat Derived Adipocytes

FIG. 22A depicts nicotinic acid and related compounds inhibitingisoproterenol induced lipolysis in rat epididymal fat derived adipocytesat a concentration of 10 μM. P-3-T represents 3-tetrazole-5-pyridine.

FIG. 22B illustrates a nicotinic acid dose-dependent inhibition ofisoproterenol induced-lipolysis in rat epididymal fat derivedadipocytes. Note the rightward shift in the dose-response curves withincreasing concentrations of nicotinic acid.

Lipolysis assays were done following the isolation of adipocytes fromrat or human. The source of fat from rats was the epididymal fat andfrom humans was either subcutaneous or omental. Cells were isolatedfollowing collagenase digestion and floatation. An elevation ofintracellular cAMP levels and concomitant activation of lipolysis viahormone sensitive lipase was accomplished using isoproterenol,forskolin, 3-isobutyl-1-methyl-xanthine (IBMX) or a combination thereofat concentrations and times determined empirically and depending on thesource of tissue. Lipolysis was allowed to continue for the desired timein the presence or absence of drug (e.g. nicotinic acid, P-3-T, etc).Data was analyzed using Excel and PrismGraph.

To show that a modulator of hRUP19 behaves similarly, an analogous assayis set up using said modulator of hRUP19. Preferred said modulator is anagonist.

To show that a modulator of hRUP38 behaves similarly, an analogous assayis set up using said modulator of hRUP38, wherein the rat is transgenicfor hRUP38. Preferred said modulator is an agonist.

To show that a modulator of hRUP11 behaves similarly, an analogous assayis set up using said modulator of hRUP11, wherein the rat is transgenicfor hRUP11. Preferred said modulator is an agonist.

Example 16 Dose-Dependent Inhibition of Isoproterenol Induced-Lipolysisin Human, Subcutaneous-Derived, Primary Adipocytes Via Nicotinic Acidand P-3-T

FIG. 23 illustrates the ability of both nicotinic acid and the relatedcompound P-3-T (3-tetrazole-5-pyridine) to inhibit isoproterenol inducedlipolysis in adipocyte primary cultures derived from human subcutaneousfat in a dose-dependant manner. The EC₅₀ value for nicotinic acid andP-3-T were 716 nM and 218 nM respectively. (Also see Example 15, supra.)

Example 17 Screening Data for Nicotinic Acid and1-Isopropyl-1H-Benzotriazole-5-Carboxylic Acid in cAMP Assays

FIG. 24 presents screening data via adenylyl cyclase assay for hRUP38.Note that nicotinic acid does not activate inhibition of forskolinstimulated cAMP hRUP38-expressing CHO cells whereas1-Isopropyl-1H-benzotriazole-5-carboxylic acid does.1-Isopropyl-1H-benzotriazole-5-carboxylic acid has no effect on CHOcells expressing either hRUP25 or hRUP19.

Example 18 Inhibition of Isoproterenol Stimulated Lipolysis 1N HumanSubcutaneous Adipocytes

Nicotinic acid (an agonist of hRUP25) and1-Isopropyl-1H-benzotriazole-5-carboxylic acid (an agonist of hRUP38;see Example 17, supra) were separately dose-dependently applied toisoproterenol (100 nM) stimulated primary human adipocytes. FIG. 25illustrates the ability of 1-Isopropyl-1H-benzotriazole-5-carboxylicacid to inhibit isoproterenol stimulated lipolysis in adipocyte primarycultures derived from human subcutaneous fat in a dose-dependent mannercomparable to that of nicotinic acid.

To show that a modulator of hRUP19 behaves similarly, an analogous assayis set up using said modulator of hRUP19. Preferred said modulator is anagonist.

To show that a modulator of hRUP11 behaves similarly, an analogous assayis set up using said modulator of hRUP11. Preferred said modulator is anagonist.

Example 19 Inhibition of Forskolin Stimulated cAMP Accumulation inhRUP38-CHO Stable Cell Line by 3-(Bromo-2-Ethoxy-Phenyl)-Acrylic Acid

FIG. 26 presents screening data via adenylyl cyclase assay for hRUP38.Note that 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid activates inhibitionof forskolin stimulated cAMP in hRUP38-expressing CHO cells but has noeffect on CHO cells expressing either hRUP25 or hRUP19. The EC₅₀ for3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid is 1.17 μM. (Also see thelegend to Example 11, supra, for details directed to stable CHOtransfectants.)

Example 20 RT-PCR Indicates that hRUP19 is Selectively Expressed 1NHuman Fat Cells

FIG. 27 presents an RT-PCR analysis of hRUP19 expression using a panelof human tissues. The analysis indicates that hRUP19 is selectivelyexpressed in fat cells. Low expression is also evident in testis,placenta, kidney and spleen.

Oligonucleotides used for PCR had the following sequences:5′-GGCCGTGGCTGATTTCCTCCTTAT-3′ (SEQ. ID. NO.:152; forward primer) and5′-AACCGGGTCGCCTTCTTCATCC-3′ (SEQ. ID. NO.:153; reverse primer).Commercially available human multiple tissue cDNA panels were used astemplates [Clontech, MTC panels Human I (#K1420-1) and Human II(K-1421-1), and human fat cell cDNA (#7128-1)]. 1 ng cDNA was used perPCR amplification. PCR was performed using Platinum PCR SuperMix (LifeTechnologies, Inc.); according to manufacturer instructions. Thefollowing cycles were used: 95° C. for 4 min; 95° C. for 1 min; 60.5° C.for 30 sec, 72° C. for 1 min, and 72° C. for 7 min; cycles 2 through 4were repeated 35 times. The resulting PCR reactions (15 μl) were loadedon a 1.5% agarose gel to analyze the RT-PCR products, and a specific 492base-pair DNA fragment representing hRUP19 was specifically amplifiedfrom cDNA of fat cell origin. Low expression was also evident in testis,placenta, kidney, and spleen.

Example 21 Northern Blot Analysis of hRUP19 Expression 1N SelectedTissues

FIG. 28 presents a Northern blot analysis of hRUP19 expression using apanel of human tissues. The analysis indicates that hRUP19 is stronglyexpressed in mammary gland, probably due to fat cell-specific expressionof hRUP19. Ad, adrenal gland; Bl, bladder, BM, bone marrow; Br, brain(whole); LN, lymph node; MG, mammary gland; Pr, prostate; Sp, spinalcord; St, stomach; Thyr, thyroid; Trch, trachea; Ut, uterus.

A pre-made blot containing Poly A+ RNA from 12 human tissues waspurchased from Clontech (Human 12-Lane II, Cat. # 7784-1). hRUP19 cDNAprobe encompassing the coding region was generated by PCR using aplasmid containing hRUP19 cDNA as template. The blot was prehybridizedwith 20 ml Clontech ExpressHyb solution (Cat. # 8015-2) for 30 minutesin a hybridization oven at 65° C., according to the manufacturer'sdirections. 25 ng of a random primer-labeled, hRUP19 cDNA probe wasadded to the prehybridization solution, and the incubation was continuedfor an additional 2 hr. The membrane was then washed according to themanufacturer's directions (Cat. # 8015-2) and exposed to film forautoradiography over a period of two days. Ad, adrenal gland; BI,bladder, BM, bone marrow; Br, brain (whole); LN, lymph node; MG, mammarygland; Pr, prostate; Sp, spinal cord; St, stomach; Thyr, thyroid; Trch,trachea; Ut, uterus.

Example 22 RUP19 Expression is Induced During Adipocyte Differentiation:Characterization of mRUP19 Expression in Mouse 3T3 Pre-Adipocytes andAdipocytes

FIG. 29 presents an analysis of RUP19 mRNA expression as a function ofadipocyte differentiation. The analysis indicates that RUP19 mRNAexpression is induced during adipocyte differentiation.

3T3 pre-adipocytes were cultured in DMEM containing 10% bovine calfserum. These cells were induced to differentiate into an adipocytephenotype using a standard protocol [Haraguchi K et al (1996) BiochemBiophys Res Comm 223:193-198; the disclosure of which is herebyincorporated by reference in its entirety]. Briefly, 1 day afterconfluence, cells were treated with DMEM containing 10% FBS, 10 μg/mlinsulin, 0.2 μg/ml dexamethasone, and 0.5 mM isobutylmethylxanthine.After 3 days, the cells were shifted to media supplemented with 10 μg/mlinsulin and 10% FBS, and 2 days later, the cells were shifted to mediacontaining 10% FBS alone. After an additional 48 hrs, total RNA wasisolated from undifferentiated or differentiated cells using RNAzolaccording to the manufacturer's directions. Separate populations ofundifferentiated and differentiated cells were subjected to stainingwith Oil Red, to confirm the induction of an adipocyte phenotype withthis protocol.

Northern blot analysis. 10 ug of total RNA from 293 cells, 3T3preadipocytes and 3T3 adipocytes was subjected to electrophoresis on a1% agarose/formaldehyde gel and transferred to a nylon membrane usingstandard protocols. The blot was hybridized to a 361 bp mRUP19 cDNAprobe and exposed to film as described in FIG. 28. The mRUP19 cDNA probecorresponds to nucleotides 5-365 of SEQ. ID. NO.:150.

RT-PCR analysis. To detect mRUP19 mRNA by RT-PCR, the following primerswere used: 5′-ACTGTGGTGGCTGTGGATAGGTA-3′ (SEQ. ID. NO.:154; forwardprimer) and 5′-GCAGATTGTGAGCTTGGCGTAGAA-3′ (SEQ. ID. NO.:155; reverseprimer). These are predicted to generate an mRUP19 product of 567 bp.The cDNA templates were prepared using a RETROscript™ First StrandSynthesis Kit for RT-PCR (Ambion, Inc., Cat. # 1710), according to themanufacturer's directions, except that duplicate reactions were done foreach input RNA, and in one of these, reverse transcriptase was excludedfrom the reaction. 3 ul of each reaction was used for PCR. The reactionconditions for the PCR were as follows: 1 cycle @ 94° C./5 min., 25cycles @ 94° C./30 sec, 59° C./30 sec, 72° C./1 min, and 1 cycle @ 72°C./5 min. The reactions were then analyzed on a 1% agarose gel. Pre-diff3T3-L1, mouse 3T3 pre-adipocytes; Post-diff 3T3-L1, differentiated 3T3adipocytes; β-TC-6, a mouse insulin-producing cell line; NIT-1, a mouseinsulin-producing cell line.

Example 23 Cart-Activated hRUP19 Inhibits Camp Production in Membranesof Transfected 293 Cells

FIG. 30 presents a CART analysis of signal transduction by hRUP19. Theanalysis indicates that CART-activated hRUP19 inhibits cAMP productionin membranes of transfected 293 cells.

Membranes were prepared as follows. 15 ug of the following expressionplasmids were each introduced into 293 cells (one 15 cm dish pertransfection) using Lipofectamine Reagent (Invitrogen, #18324-020)according to the manufacturer's instructions: pCMV-MCS (empty CMVexpression plasmid), pCMV-hRUP19, pCMV-hRUP19-CART (same as pCMV-hRUP19,except that codon 219 has been converted from threonine to lysine).After 48 hours, a crude membrane preparation was prepared using standardprotocols. Briefly, cells were washed with ice cold PBS, removed fromthe plate by scraping in the presence of a hypotonic Tris/EDTA buffer,fragmented using a pre-chilled dounce homogenizer, spun at low speed topellet nuclei and intact cells, and finally, the supernatant issubjected to centrifugation at 20,000 rpm in a Beckman Avanti J-20centrifuge. The membrane pellet is then resuspended at a proteinconcentration of 1 mg/ml for use in a membrane cyclase assay. Themembrane cyclase assay was carried out as per the manufacturer'srecommendation using an Adenylyl Cyclase Activation FlashPlate Assay Kit(Perkin Elmer Life Sciences, Inc., #SMP004B).

Example 24 Summary: hRUP25, mRUP25, rRUP25, hRUP38, hRUP19, mRUP19,rRUP19, AND hRUP11

TABLE M Disclosed Gi-Coupled Nicotinic Acid (Lowers the ReceptorExpression by Level of Shown to Inhibit Sub-Family Adipocytes orIntracellular Intracellular GPCRs Adipose cAMP) Lipolysis Agonist hRUP25yes yes yes nicotinic acid; (−)-nicotine; see FIG. 21; (5-hydroxy-1-methyl-3-propyl-1H- pyrazol-4-yl)-pyridin- 3-yl-methanone mRUP25 yes yesn.d. nicotinic acid rRUP25 yes yes yes nicotinic acid hRUP38 yes yes yes1-Isopropyl-1H- benzotriazole-5- carboxylic acid; 3-(5-Bromo-2-ethoxy-phenyl)-acrylic acid hRUP11 n.d. yes n.d. n.d. hRUP19 yes yes n.d. n.d.mRUP19 yes n.d. n.d. n.d. rRUP19 n.d. n.d. n.d. n.d.n.d.: not displayed

Example 25 Rodent Diabetes Models

Rodent models of type 2 diabetes associated with obesity and insulinresistance have been developed. Genetic models such as db/db and ob/ob[see Diabetes (1982) 31:1-6] in mice and fa/fa in zucker rats have beendeveloped for understanding the pathophysiology of disease and fortesting candidate therapeutic compounds [Diabetes (1983) 32:830-838;Annu Rep Sankyo Res Lab (1994) 46:1-57]. The homozygous animals, C57BL/KsJ-db/db mice developed by Jackson Laboratory are obese,hyperglycemic, hyperinsulinemic and insulin resistant [J Clin Invest(1990) 85:962-967], whereas heterozygotes are lean and normoglycemic. Inthe db/db model, mice progressively develop insulinopenia with age, afeature commonly observed in late stages of human type 2 diabetes whensugar levels are insufficiently controlled. Since this model resemblesthat of human type 2 diabetes, the compounds of the present inventionare tested for activities including, but not limited to, lowering ofplasma glucose and triglycerides. Zucker (fa/fa) rats are severelyobese, hyperinsulinemic, and insulin resistant {Coleman, Diabetes (1982)31:1; E Shafrir in Diabetes Mellitus, H Rifkin and D Porte, Jr, Eds[Elsevier Science Publishing Co, New York, ed. 4, (1990), pp. 299-340]},and the fa/fa mutation may be the rat equivalent of the murine dbmutation [Friedman et al, Cell (1992) 69:217-220; Truett et al, ProcNatl Acad Sci USA (1991) 88:7806]. Tubby (tub/tub) mice arecharacterized by obesity, moderate insulin resistance andhyperinsulinemia without significant hyperglycemia [Coleman et al,Heredity (1990) 81:424].

The present invention encompasses the use of compounds of the inventionfor reducing the insulin resistance and hyperglycemia in any or all ofthe above rodent diabetes models, in humans with type 2 diabetes orother preferred metabolic-related disorders or disorders of lipidmetabolism described previously, or in models based on other mammals.Plasma glucose and insulin levels will be tested, as well as otherfactors including, but not limited to, plasma free fatty acids andtriglycerides.

In Vivo Assay for Anti-Hyperglycemic Activity of Compounds of theInvention

Genetically altered obese diabetic mice (db/db) (male, 7-9 weeks old)are housed (7-9 mice/cage) under standard laboratory conditions at 22°C. and 50% relative humidity, and maintained on a diet of Purina rodentchow and water ad libitum. Prior to treatment, blood is collected fromthe tail vein of each animal and blood glucose concentrations aredetermined using One Touch Basic Glucose Monitor System (Lifescan). Micethat have plasma glucose levels between 250 to 500 mg/dl are used. Eachtreatment group consists of seven mice that are distributed so that themean glucose levels are equivalent in each group at the start of thestudy. db/db mice are dosed by micro-osmotic pumps, inserted usingisoflurane anesthesia, to provide compounds of the invention, saline, oran irrelevant compound to the mice subcutaneously (s.c.). Blood issampled from the tail vein at intervals thereafter and analyzed forblood glucose concentrations. Significant differences between groups(comparing compounds of the invention to saline-treated) are evaluatedusing Student t-test.

The foregoing is provided by way of illustration and not limitation.Other illustrative rodent models for type 2 diabetes have been described[Moller D E, Nature (2001) 414:821-7 and references therein; and Reed MJ et al., Diabetes, Obesity and Metabolism (1999) 1:75-86 and referencetherein; the disclosure of each of which is hereby incorporated byreference in its entirety].

Example 26 Mouse Atherosclerosis Model

Adiponectin-deficient mice generated through knocking out theadiponectin gene have been shown to be predisposed to atherosclerosisand to be insulin resistant. The mice are also a suitable model forischemic heart disease [Matsuda, M et al. J Biol Chem (2002) July, andreferences cited therein, the disclosures of which are incorporatedherein by reference in their entirety].

Adiponectin knockout mice are housed (7-9 mice/cage) under standardlaboratory conditions at 22° C. and 50% relative humidity. The mice aredosed by micro-osmotic pumps, inserted using isoflurane anesthesia, toprovide compounds of the invention, saline, or an irrelevant compound tothe mice subcutaneously (s.c.). Neointimal thickening and ischemic heartdisease are determined for different groups of mice sacrificed atdifferent time intervals. Significant differences between groups(comparing compounds of the invention to saline-treated) are evaluatedusing Student t-test.

The foregoing mouse model of atherosclerosis is provided by way ofillustration and not limitation. By way of further example,Apolipoprotein E-deficient mice have also been shown to be predisposedto atherosclerosis [Plump A S et al., Cell (1992) 71:343-353; thedisclosure of which is hereby incorporated by reference in itsentirety].

A preferred model is that of diet-induced atherosclerosis in C57BL/6Jmice, an inbred strain known to be susceptible to diet-inducedatherosclerotic lesion formation. This model is well known to thosepersons of ordinary skill in the art [Kamada N et al., J AtherosclerThromb (2001) 8:1-6; Garber D W et al., J Lipid Res (2001) 42:545-52;Smith J D et al., J Intern Med (1997) 242:99-109; the disclosure of eachof which is hereby incorporated by reference in its entirety].

Example 27 Transgenic Mouse/Rat

hRUP38

The present invention also provides methods and compositions for thegeneration of mice and rats that express hRUP38 recombinant humanantilipolytic GPCR polyeptide of the invention.

Methods of making transgenic animals such as mice and rats are wellknown to those of ordinary skill in the art, and any such method can beused in the present invention. Briefly, transgenic mammals can beproduced, e.g., by transfecting a pluripotential stem cell such as an EScell with a polynucleotide encoding hRUP38 polypeptide of the invention.Successfully transformed ES cells can then be introduced into an earlystage embryo that is then implanted into the uterus of a mammal of thesame species. In certain cases, the transformed (“transgenic”) cellswill comprise part of the germ line of the resulting animal and adultanimals comprising the transgenic cells in the germ line can then bemated to other animals, thereby eventually producing a population oftransgenic animals that have the transgene in each of their cells andthat can stably transmit the transgene to each of their offspring. Othermethods of introducing the polynucleotide can be used, for exampleintroducing the polynucleotide encoding hRUP38 polypeptide of theinvention into a fertilized egg or early stage embryo viamicroinjection. Alternatively, the transgene may be introduced into ananimal by infection of zygotes with a retrovirus containing thetransgene [Jaenisch, R, Proc Natl Acad Sci USA (1976) 73:1260-4].Methods of making transgenic mammals are described, e.g., in Wall etal., J Cell Biochem (1992) 49:113-20; Hogan et al., in Manipulating theMouse Embryo. A Laboratory Manual. (1986) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; in WO 91/08216; or in U.S. Pat. No.4,736,866; all of which disclosures are hereby incorporated by referencein their entirety.

In preferred embodiments, expression of said gene is placed under thecontrol of an essentially adipocyte specific promoter. In furtherpreferred embodiments, said essentially adipocyte specific promoter isadiponectin gene promoter [Das, K et al., Biochem Biophys Res Commun(2001) 280:1120-9; Barth, N et al., Diabetologia (2002) 45:1425-1433;the disclosures of which are hereby incorporated by reference in itsentirety]. In other further preferred embodiments, said essentiallyadipocyte specific promoter is resistin gene promoter [Hartman, H B etal. J Biol Chem (2002) 277:19754-61, which disclosure is herebyincorporated by reference in its entirety]. In other preferredembodiments, said essentially adipocyte specific promoter is aP2[Felmer, R et al., J Endocrinol (2002) 175:487-498; the disclosure ofwhich is hereby incorporated by reference in its entirety]. In otherfurther preferred embodiments, expression of said gene is kept under thecontrol of its endogenous promoter.

hRUP11

The present invention also provides methods and compositions for thegeneration of mice and rats that express hRUP11 recombinant humanantilipolytic GPCR polyeptide of the invention.

Methods of making transgenic animals such as mice and rats are wellknown to those of ordinary skill in the art, and any such method can beused in the present invention. Briefly, transgenic mammals can beproduced, e.g., by transfecting a pluripotential stem cell such as an EScell with a polynucleotide encoding hRUP11 polypeptide of the invention.Successfully transformed ES cells can then be introduced into an earlystage embryo that is then implanted into the uterus of a mammal of thesame species. In certain cases, the transformed (“transgenic”) cellswill comprise part of the germ line of the resulting animal and adultanimals comprising the transgenic cells in the germ line can then bemated to other animals, thereby eventually producing a population oftransgenic animals that have the transgene in each of their cells andthat can stably transmit the transgene to each of their offspring. Othermethods of introducing the polynucleotide can be used, for exampleintroducing the polynucleotide encoding hRUP11 polypeptide of theinvention into a fertilized egg or early stage embryo viamicroinjection. Alternatively, the transgene may be introduced into ananimal by infection of zygotes with a retrovirus containing thetransgene [Jaenisch, R, Proc Natl Acad Sci USA (1976) 73:1260-4].Methods of making transgenic mammals are described, e.g., in Wall etal., J Cell Biochem (1992) 49:113-20; Hogan et al., in Manipulating theMouse Embryo. A Laboratory Manual. (1986) Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; in WO 91/08216; or in U.S. Pat. No.4,736,866; all of which disclosures are hereby incorporated by referencein their entirety.

In preferred embodiments, expression of said gene is placed under thecontrol of an adipocyte specific promoter. In further preferredembodiments, said adipocyte specific promoter is adiponectin genepromoter [Das, K et al., Biochem Biophys Res Commun (2001) 280:1120-9;Barth, N et al., Diabetologia (2002) 45:1425-1433; the disclosures ofwhich are hereby incorporated by reference in its entirety]. In otherfurther preferred embodiments, said adipocyte specific promoter isresistin gene promoter [Hartman, H B et al. J Biol Chem (2002)277:19754-61, which disclosure is hereby incorporated by reference inits entirety]. In other preferred embodiments, said adipocyte specificpromoter is aP2 [Felmer, R et al., J Endocrinol (2002) 175:487-498; thedisclosure of which is hereby incorporated by reference in itsentirety]. In other further preferred embodiments, expression of saidgene is kept under the control of its endogenous promoter.

Example 28 Knockout Mouse/Rat

Mouse

RUP25

A preferred DNA construct will comprise, from 5′-end to 3′-end: (a) afirst nucleotide sequence that is comprised in the mRUP25 genomicsequence; (b) a nucleotide sequence comprising a positive selectionmarker, such as the marker for neomycin resistance (neo); and (c) asecond nucleotide sequence that is comprised in the mRUP25 genomicsequence and is located on the genome downstream of the first mRUP25nucleotide sequence (a). mRUP25 genomic sequence will be isolated usingmethods well known to those of ordinary skill in the art (Maniatis T etal., Molecular Cloning: A Laboratory Manual (1989) Cold Spring HarborLaboratory; the disclosure of which is hereby incorporated by referencein its entirety).

In preferred embodiments, this DNA construct also comprises a negativeselection marker located upstream the nucleotide sequence (a) ordownstream the nucleotide sequence (c). Preferably, the negativeselection marker comprises the thymidine kinase (tk) gene [Thomas etal., Cell (1986) 44:419-28], the hygromycin beta gene [Te Riele et al.,Nature (1990) 348:649-51], the hprt gene [Van der Lugt et al., Gene(1991) 105:263-7; Reid et al., Proc Natl Acad Sci USA (1990)87:4299-4303] or the Diptheria toxin A fragment (Dt-A) gene [Nada etal., Cell (1993) 73:1125-35; Yagi et al., Proc Natl Acad Sci USA (1990)87:9918-9922], which disclosures are hereby incorporated by reference intheir entireties. Preferably, the positive selection marker is locatedwithin an mRUP25 exon sequence so as to interrupt the sequence encodingan mRUP25 polypeptide. These replacement vectors are described, forexample, by Thomas et al., Cell (1986) 44:419-28; Thomas et al., Cell(1987) 51:503-12; Mansour et al., Nature (1988) 336:348-52; Koller etal., Annu Rev Immunol (1992) 10:705-30; and U.S. Pat. No. 5,631,153;which disclosures are hereby incorporated by reference in theirentireties.

The first and second nucleotide sequences (a) and (c) may beindifferently located within an mRUP25 regulatory sequence, an intronicsequence, an exon sequence or a sequence containing both regulatoryand/or intronic and/or exon sequences. The size of the nucleotidesequences (a) and (c) ranges from 1 to 50 kb, preferably from 1 to 10kb, more preferably from 2 to 6 kb, and most preferably from 2 to 4 kb.

Methods of making a knockout mouse are well known to those of ordinaryskill in the art and have been used to successfully inactivate a widerange of genes.

RUP19

A preferred DNA construct will comprise, from 5′-end to 3′-end: (a) afirst nucleotide sequence that is comprised in the mRUP19 genomicsequence; (b) a nucleotide sequence comprising a positive selectionmarker, such as the marker for neomycin resistance (neo); and (c) asecond nucleotide sequence that is comprised in the mRUP19 genomicsequence and is located on the genome downstream of the first mRUP19nucleotide sequence (a). mRUP19 genomic sequence will be isolated usingmethods well known to those of ordinary skill in the art (Maniatis T etal., Molecular Cloning: A Laboratory Manual (1989) Cold Spring HarborLaboratory; the disclosure of which is hereby incorporated by referencein its entirety).

In preferred embodiments, this DNA construct also comprises a negativeselection marker located upstream the nucleotide sequence (a) ordownstream the nucleotide sequence (c). Preferably, the negativeselection marker comprises the thymidine kinase (tk) gene [Thomas etal., Cell (1986) 44:419-28], the hygromycin beta gene [Te Riele et al.,Nature (1990) 348:649-51], the hprt gene [Van der Lugt et al., Gene(1991) 105:263-7; Reid et al., Proc Natl Acad Sci USA (1990)87:42994303] or the Diptheria toxin A fragment (Dt-A) gene [Nada et al.,Cell (1993) 73:1125-35; Yagi et al., Proc Natl Acad Sci USA (1990)87:9918-9922], which disclosures are hereby incorporated by reference intheir entireties. Preferably, the positive selection marker is locatedwithin an mRUP19 exon sequence so as to interrupt the sequence encodingan mRUP19 polypeptide. These replacement vectors are described, forexample, by Thomas et al., Cell (1986) 44:419-28; Thomas et al., Cell(1987) 51:503-12; Mansour et al., Nature (1988) 336:348-52; Koller etal., Annu Rev Immunol (1992) 10:705-30; and U.S. Pat. No. 5,631,153;which disclosures are hereby incorporated by reference in theirentireties.

The first and second nucleotide sequences (a) and (c) may beindifferently located within an mRUP19 regulatory sequence, an intronicsequence, an exon sequence or a sequence containing both regulatoryand/or intronic and/or exon sequences. The size of the nucleotidesequences (a) and (c) ranges from 1 to 50 kb, preferably from 1 to 10kb, more preferably from 2 to 6 kb, and most preferably from 2 to 4 kb.

Methods of making a knockout mouse are well known to those of ordinaryskill in the art and have been used to successfully inactivate a widerange of genes.

Rat

RUP25

Gene targeting technology for the rat is less robust than that for themouse and is an area of active interest. One approach will be toinactivate rRUP25 gene in rat embryonic stem cell (ESC)-like cells andthen inject cells with inactivated rRUP25 gene into rat blastocystsgenerated after fusion of two-cell embryos [Krivokharchenko et al., MolReprod Dev (2002) 61:460-5].

An alternative approach will be to inactivate rRUP25 gene in ratESC-like cells and then transfer the nucleus of the rat ESC-like cellshaving inactivated rRUP25 gene into enucleated oocytes [Sato K et al.,Hum Cell (2001) 14:301-4; Wakayama and Yanagimachi, Semin Cell Dev Biol(1999) 10:253-8; Hochedlinger and Jaenisch, Nature (2002) 415:1035-8;Yanagimachi, Mol Cell Endocrinol (2002) 187:241-8; the disclosures ofwhich are incorporated herein by reference in their entireties].

rRUP25 genomic sequence can be isolated using methods well known tothose of ordinary skill in the art (Maniatis T et al., MolecularCloning: A Laboratory Manual (1989) Cold Spring Harbor Laboratory; thedisclosure of which is hereby incorporated by reference in itsentirety).

RUP19

Gene targeting technology for the rat is less robust than that for themouse and is an area of active interest. One approach will be toinactivate rRUP19 gene in rat embryonic stem cell (ESC)-like cells andthen inject cells with inactivated rRUP19 gene into rat blastocystsgenerated after fusion of two-cell embryos [Krivokharchenko et al., MolReprod Dev (2002) 61:460-5].

An alternative approach will be to inactivate rRUP19 gene in ratESC-like cells and then transfer the nucleus of the rat ESC-like cellshaving inactivated rRUP25 gene into enucleated oocytes [Sato K et al.,Hum Cell (2001) 14:3014; Wakayama and Yanagimachi, Semin Cell Dev Biol(1999) 10:253-8; Hochedlinger and Jaenisch, Nature (2002) 415:1035-8;Yanagimachi, Mol Cell Endocrinol (2002) 187:241-8; the disclosures ofwhich are incorporated herein by reference in their entireties].

rRUP19 genomic sequence can be isolated using methods well known tothose of ordinary skill in the art (Maniatis T et al., MolecularCloning: A Laboratory Manual (1989) Cold Spring Harbor Laboratory; thedisclosure of which is hereby incorporated by reference in itsentirety).

CRE-LOXP System

These new DNA constructs make use of the site specific recombinationsystem of the P1 phage. The P1 phage possesses a recombinase called Crethat interacts with a 34 base pair loxP site. The loxP site is composedof two palindormic sequences of 13 bp separated by an 8 bp conservedsequence [Hoess R H et al, Nucleic Acids Res (1986) 14:2287-300; whichdisclosure is hereby incorporated by reference in its entirety]. Therecombination by the Cre enzyme between two loxP sites having anidentical orientation leads to the deletion of the DNA fragment.

The Cre-loxP system used in combination with a homologous recombinationtechnique has been first described by Gu et al. [Gu H et al., Cell(1993) 73:1155-64; Gu H et al., Science (1994) 265:103-6; whichdisclosures are hereby incorporated by reference in their entirety].Briefly, a nucleotide sequence of interest to be inserted in a targetedlocation of the genome harbors at least two loxP sites in the sameorientation and located at the respective ends of a nucleotide sequenceto be excised from the recombinant genome. The excision event requiresthe presence of the recombinase (Cre) enzyme within the nucleus of therecombinant cell host. The recombinase enzyme may be brought at thedesired time either by (a) incubating the recombinant cell hosts in aculture medium containing this enzyme, by injecting the Cre enzymedirectly into the desired cell, such as by lipofection of the enzymeinto the cells, such as described by Baubonis et al. [Baubonis W andSauer B, Nucleic Acids Res (1993) 21:2025-9; which disclosure is herebyincorporated by reference in its entirety]; (b) transfecting the cellhost with a vector comprising the Cre Coding sequence operably linked toa promoter functional in the recombinant cell host, which promoter beingoptionally inducible, said vector being introduced in the recombinantcell host, such as described by Gu et al. [Gu H et al., Cell (1993)73:1155-64; which disclosure is hereby incorporated by reference in itsentirety] and Sauer et al. [Sauer B and Henderson N, Proc Natl Acad SciUSA (1988) 85:5166-70; which disclosure is hereby incorporated byreference in its entirety]; (c) introducing into the genome of the cellhost a polynucleotide comprising the Cre coding sequence operably linkedto a promoter functional in the recombinant cell host, which promoter isoptionally inducible, and said polynucleotide being inserted in thegenome of the cell host either by a random insertion event or anhomologous recombination event, such as described by Gu et al. [Gu H etal., Science (1994) 265:103-6; which disclosure is hereby incorporatedby reference in its entirety].

Vectors and methods using the Cre-loxP system are describe by Zou et al.(1994), which disclosure is hereby incorporated by reference in itsentirety.

In preferred embodiments of the invention, Cre is introduced into thegenome of the cell host by strategy (c) above, wherein said promoter isessentially adipocyte specific and leads to essentially adipocytespecific knockout of (loxP-flanked) mRUP25 or mRUP19 in the mouse orrRUP25 or rRUP19 in the rat. In some embodiments, said essentiallyadipocyte specific promoter is adiponectin gene promoter [Das, K et al.,Biochem Biophys Res Commun (2001) 280:1120-9; Barth, N et al.,Diabetologia (2002) 45:1425-1433; the disclosures of which are herebyincorporated by reference in its entirety]. In some embodiments, saidessentially adipocyte specific promoter is resistin gene promoter[Hartman, H B et al. J Biol Chem (2002) 277:19754-61, which disclosureis hereby incorporated by reference in its entirety]. In someembodiments, said essentially adipocyte specific promoter is aP2[Felmer, R et al., J Endocrinol (2002) 175:487-498; the disclosure ofwhich is hereby incorporated by reference in its entirety]. Methods ofconstructing a lineage-specific knockout are well known to persons ofordinary skill in the art, as illustrated but not intended to be limitedby: Kuhn R and Torres R M, Methods Mol Biol (2002) 180:175-204; Sauer B,Methods (1998) 14:381-92; Gutstein D E et al., Circulation Research(2001) 88:333; Minamino T et al., Circulation Research (2001) 88:587;and Bex A et al., J Urol (2002) 168:2641-2644; the disclosure of each ofwhich is hereby incorporated by reference in its entirety.

Example 29 Fluorometric Imaging Plate Reader (FLIPR) Assay for theMeasurement of Intracellular Calcium Concentration

Target Receptor (experimental) and pCMV (negative control) stablytransfected cells from respective clonal lines are seeded intopoly-D-lysine pretreated 96-well plates (Becton-Dickinson, #356640) at5.5×10⁴ cells/well with complete culture medium (DMEM with 10% FBS, 2 mML-glutamine, 1 mM sodium pyruvate) for assay the next day. To prepareFluo-4-AM (Molecular Probe, #F14202) incubation buffer stock, 1 mgFluo4-AM is dissolved in 467 μl DMSO and 467 μl Pluoronic acid(Molecular Probe, #P3000) to give a 1 mM stock solution that can bestored at −20° C. for a month. Fluo4-AM is a fluorescent calciumindicator dye.

Candidate compounds are prepared in wash buffer (1×HBSS/2.5 mMProbenicid/20 mM HEPES at pH 7.4).

At the time of assay, culture medium is removed from the wells and thecells are loaded with 100 μl of 4 μM Fluo4-AM/2.5 mM Probenicid (Sigma,#P8761)/20 mM HEPES/complete medium at pH 7.4. Incubation at 37° C./5%CO₂ is allowed to proceed for 60 min.

After the 1 hr incubation, the Fluo4-AM incubation buffer is removed andthe cells are washed 2× with 100 μL wash buffer. In each well is left100 μl wash buffer. The plate is returned to the incubator at 37° C./5%CO₂ for 60 min.

FLIPR (Fluorometric Imaging Plate Reader; Molecular Device) isprogrammed to add 50 μl candidate compound on the 30^(th) second and torecord transient changes in intracellular calcium concentration ([Ca²⁺])evoked by the candidate compound for another 150 seconds. Totalfluorescence change counts are used to determine agonist activity usingthe FLIPR software. The instrument software normalizes the fluorescentreading to give equivalent initial readings at zero.

In some embodiments, the cells comprising Target Receptor furthercomprise promiscuous G alpha 15/16 or the chimeric Gq/Gi alpha unit.

Although the foregoing provides a FLIPR assay for agonist activity usingstably transfected cells, a person of ordinary skill in the art wouldreadily be able to modify the assay in order to characterize antagonistactivity. Said person of ordinary skill in the art would also readilyappreciate that, alternatively, transiently transfected cells could beused.

Example 30 IN VIVO Pig Model of HDL-Cholesterol and Atherosclerosis

The utility of a modulator of the present invention as a medical agentin the prevention or treatment of a high totalcholesterol/HDL-cholesterol ratio and conditions relating thereto isdemonstrated, without limitation, by the activity of the modulator inlowering the ratio of total cholesterol to HDL-cholesterol, in elevatingHDL-cholesterol, or in protection from atherosclerosis in an in vivo pigmodel. Pigs are used as an animal model because they reflect humanphysiology, especially lipid metabolism, more closely than most otheranimal models. An illustrative in vivo pig model not intended to belimiting is presented here.

Yorkshire albino pigs (body weight 25.5±4 kg) are fed a saturated fattyacid rich and cholesterol rich (SFA-CHO) diet during 50 days (1 kg chow35 kg⁻¹ pig weight), composed of standard chow supplemented with 2%cholesterol and 20% beef tallow [Royo T et al., European Journal ofClinical Investigation (2000) 30:843-52; which disclosure is herebyincorporated by reference in its entirety]. Saturated to unsaturatedfatty acid ratio is modified from 0.6 in normal pig chow to 1.12 in theSFA-CHO diet. Animals are divided into two groups, one group (n=8) fedwith the SFA-CHO diet and treated with placebo and one group (n=8) fedwith the SFA-CHO diet and treated with the modulator (3.0 mg kg⁻¹).Control animals are fed a standard chow for a period of 50 days. Bloodsamples are collected at baseline (2 days after the reception of theanimals), and 50 days after the initiation of the diet. Blood lipids areanalyzed. The animals are sacrificed and necropsied.

Alternatively, the foregoing analysis comprises a plurality of groupseach treated with a different dose of the modulator. Preferred saiddoses are selected from the group consisting of: 0.1 mg kg⁻¹, 0.3 mgkg⁻¹, 1.0 mg kg⁻¹, 3.0 mg kg⁻¹, 10 mg kg⁻¹, 30 mg kg⁻¹ and 100 mg kg⁻¹.Alternatively, the foregoing analysis is carried out at a plurality oftimepoints. Preferred said timepoints are selected from the groupconsisting of 10 weeks, 20 weeks, 30 weeks, 40 weeks, and 50 weeks.

HDL-Cholesterol

Blood is collected in trisodium citrate (3.8%, 1:10). Plasma is obtainedafter centrifugation (1200 g 15 min) and immediately processed. Totalcholesterol, HDL-cholesterol, and LDL-cholesterol are measured using theautomatic analyzer Kodak Ektachem DT System (Eastman Kodak Company,Rochester, N.Y., USA). Samples with value parameters above the range arediluted with the solution supplied by the manufacturer and thenre-analyzed. The total cholesterol/HDL-cholesterol ratio is determined.Comparison is made of the level of HDL-cholesterol between groups.Comparison is made of the total cholesterol/HDL-cholesterol ratiobetween groups.

Elevation of HDL-cholesterol or reduction of the totalcholesterol/HDL-cholesterol ratio on administration of the modulator istaken as indicative of the modulator having the aforesaid utility.

Atherosclerosis

The thoracic and abdominal aortas are removed intact, openedlongitudinally along the ventral surface, and fixed in neutral-bufferedformalin after excision of samples from standard sites in the thoracicand abdominal aorta for histological examination and lipid compositionand synthesis studies. After fixation, the whole aortas are stained withSudan IV and pinned out flat, and digital images are obtained with a TVcamera connected to a computerized image analysis system (Image ProPlus; Media Cybernetics, Silver Spring, Md.) to determine the percentageof aortic surface involved with atherosclerotic lesions [Gerrity R G etal, Diabetes (2001) 50:1654-65; Cornhill J F et al, Arteriosclerosis,Thrombosis, and Vascular Biology (1985) 5:415-26; which disclosures arehereby incorporated by reference in their entirety]. Comparison is madebetween groups of the percentage of aortic surface involved withatherosclerotic lesions.

Reduction of the percentage of aortic surface involved withatherosclerotic lesions on administration of the modulator is taken asindicative of the modulator having the aforesaid utility.

Plasma Free Fatty Acids

It would be readily apparent to anyone of ordinary skill in the art thatthe foregoing in vivo pig model is easily modified in order to address,without limitation, the activity of the modulator in lowering plasmafree fatty acids.

Example 31 Measurement of Plasma Free Fatty Acids (FFA) in RatsAdministered Niacin

Catheters were surgically implanted into the jugular veins of maleSprague Dawley rats. The following day rats were deprived of food andapproximately 16 hours later were given interperitoneal (IP) injectionsof either vehicle, or niacin [NA] at 15 mg/kg, 30 mg/kg or 45 mg/kg bodyweight. Blood was drawn (˜200 ml) at the indicated time points andplasma was isolated following centrifugation. Plasma FFA were thenmeasured via the NEFA C kit according to manufacturer specifications(Wako Chemicals USA, Inc). All three concentrations of niacinsignificantly decrease plasma FFA levels. [See, FIG. 32.] * Indicates astatistical increase in plasma FFA versus vehicle control indicating arebound effect in the 45 mg/kg niacin treated rats at 3 hr (i.e., plasmaFFA levels go beyond basal levels following inhibition).

By way of illustration and not limitation, said rat model has utility asan in vivo animal model for modulators of RUP25 provided by theinvention. By way of illustration and not limitation, said rat modelalso has utility as an in vivo animal model for modulators of RUP19provided by the invention. By way of illustration and not limitation,hRUP38 and hRUP11 transgenic rats provided by the invention have utilityas in vivo animal models for modulators of hRUP38 and hRUP11 provided bythe invention.

To show that a modulator of hRUP25 other than niacin behaves similarly,an analogous assay is set up using said modulator of hRUP25. Preferredsaid modulator is an agonist.

To show that a modulator of hRUP38 behaves similarly, an analogous assayis set up using said modulator of hRUP38, wherein the rat is transgenicfor hRUP38. Preferred said modulator is an agonist.

To show that a modulator of hRUP19 behaves similarly, an analogous assayis set up using said modulator of hRUP19. Preferred said modulator is anagonist.

To show that a modulator of hRUP11 behaves similarly, an analogous assayis set up using said modulator of hRUP11, wherein the rat is transgenicfor hRUP11. Preferred said modulator is an agonist.

Example 32 Preparation of Non-Endogenous, Enhanced-for-Agonist [EFA-]GPCRS

Constitutive activity of a GPCR reduces the available window foridentification of an agonist of the GPCR, where said window is takenhere to be the difference in assay readout between the GPCR in theabsence of agonist and the GPCR in the presence of a known agonist. Amutant of a constitutively active GPCR that is less constitutivelyactive but comparably or more responsive to said known agonist[Enhanced-for-Agonist GPCR; EFA-GPCR] would have novel utility for theidentification of modulators of said GPCR, particularly agonists.

EFA-GPCR is disclosed herewith as a mutant GPCR polypeptide thatconsists of 1, 2, 3, 4, or 5 amino acid substitutions, deletions, orinsertions relative to the amino acid sequence of an endogenous GPCRpolypeptide having constitutive activity, wherein the agonist screeningwindow of the mutant GPCR is expanded by greater than 20%, greater than25%, greater than 30%, greater than 31%, greater than 32%, greater than33%, greater than 34%, greater than 35%, greater than 36%, greater than37%, greater than 38%, greater than 39%, or greater than 40% relative tothat of said endogenous GPCR.

As the GPCRs of the invention are constitutively active to a significantdegree, an EFA version of said GPCRs would have novel utility inscreening for modulators of said GPCRs, particularly agonists.

Herewith Applicant discloses EFA-hRUP25 “hRUP25-S91” polynucleotide ofSEQ. ID. NO:158 and the encoded polypeptide of SEQ. ID. NO.:159.EFA-hRUP25 polypeptide “hRUP25-S91” differs from endogenous hRUP25polypeptide of SEQ. ID. NO.:36 through substitution of the tryptophan atamino acid position 91 with serine. Mutagenesis was performed usingQuickChange Site-Directed Mutagenesis Kit (Stratagene) according to themanufacturer's instructions. The mutagenesis primers had the followingsequences:

Sense primer: CTATGTGAGGCGTTCAGACTGGAAGTTG (SEQ. ID. NO.:160;

Antisense primer: CAAACTTCCAGTCTGAACGCCTCACATAG (SEQ. ID. NO.:161.

Activity of EFA-hRUP25 “hRUP25-S91” polypeptide is presented in FIG. 33.The window for agonist screening is 44% larger for EFA-hRUP25 (433-210pmol cAMP/mg protein) relative to that of endogenous hRUP25 (329-174pmol cAMP/mg protein). {[(433−210=223)/(329−174=155)]=1.44≡144%.}

The invention relates in part to an isolated EFA-hRUP25 polypeptidecomprising the amino acid sequence of SEQ. ID. NO.:159 and to isolatedpolynucleotide encoding said EFA-hRUP25 polypeptide. A preferredEFA-hRUP25 polynucleotide has the nucleotide sequence of SEQ. ID.NO.:158.

The invention further relates in part to isolated EFA-hRUP25 polypeptidecomprising an amino acid sequence consisting of 1, 2, 3, or 4 amino acidsubstitutions, deletions, or insertions relative to the amino acidsequence of SEQ. ID. NO.:36 in addition to the substitution of serinefor tryptophan at amino acid position 91, as well as to isolatedpolynucleotide encoding said EFA-hRUP25 polypeptide.

The invention also relates in part to a method of using a polypeptidecomprising an EFA-hRUP25 amino acid sequence to identify a modulator ofEFA-hRUP25. The invention also relates to a method of using apolypeptide comprising an EFA-hRUP25 amino acid sequence to identify amodulator of lipolysis. Preferred said modulator of EFA-hRUP25 is anagonist.

Other embodiments encompass EFA-mRUP25 and EFA-rRUP25 polypeptide andpolynucleotide. Also preferred is a method of using EFA-mRUP25 orEFA-rRUP25 to identify a modulator of EFA-mRUP25 or EFA-rRUP25. Alsopreferred is a method of using EFA-mRUP25 or EFA-rRUP25 to identify amodulator of lipolysis. Preferred said modulator of EFA-mRUP25 orEFA-rRUP25 is an agonist.

Other embodiments encompass EFA-RUP38 polypeptide and polynucleotide.Also preferred is a method of using EFA-RUP38 to identify a modulator ofEFA-RUP38. Also preferred is a method of using EFA-RUP38 to identify amodulator of lipolysis. Preferred said modulator of EFA-RUP38 is anagonist.

Other embodiments encompass EFA-hRUP19 polypeptide and polynucleotide.Also preferred is a method of using EFA-hRUP19 to identify a modulatorof EFA-hRUP19. Also preferred is a method of using EFA-hRUP19 toidentify a modulator of lipolysis. Preferred said modulator ofEFA-hRUP19 is an agonist.

Other embodiments encompass EFA-mRUP19 and EFA-rRUP19 polypeptide andpolynucleotide. Also preferred is a method of using EFA-mRUP19 orEFA-rRUP19 to identify a modulator of EFA-mRUP19 or EFA-rRUP19. Alsopreferred is a method of using EFA-mRUP19 or EFA-rRUP19 to identify amodulator of lipolysis. Preferred said modulator of EFA-mRUP19 orEFA-rRUP19 is an agonist.

Other embodiments encompass EFA-RUP11 polypeptide and polynucleotide.Also preferred is a method of using EFA-RUP11 to identify a modulator ofEFA-RUP11. Also preferred is a method of using EFA-RUP11 to identify amodulator of lipolysis. Preferred said modulator of RUP11 is an agonist.

The invention also provides for a method of making an EFA mutant of anendogenous GPCR polypeptide having constitutive activity, comprising thesteps of:

(a) introducing 1, 2, 3, 4, or 5 substitutions, insertions, or deletionsinto the amino acid sequence of the endogenous GPCR polypeptide;

(b) measuring the activity of the mutant GPCR of (a) in the absence ofagonist and in the presence of a known agonist;

(c) measuring the activity of the endogenous GPCR in the absence ofagonist and in the presence of said known agonist; and

(d) comparing (b) and (c);

wherein a determination that the agonist screening window of (b) isgreater than 20%, greater than 25%, greater than 30%, greater than 31%,greater than 32%, greater than 33%, greater than 34%, greater than 35%,greater than 36%, greater than 37%, greater than 38%, greater than 39%,or greater than 40% than that of (c) identifies the mutant resultingfrom (a) to be an EFA mutant of the endogenous GPCR.

In some embodiments, the agonist screening window of (b) is greater than20% than that of (c).

In some preferred embodiments, said known agonist is nicotinic acid.

Methods of carrying out site-specific mutagenesis are well known tothose of ordinary skill in the art. [See, e.g, in Maniatis T et al.,Molecular Cloning: A Laboratory Manual (1989) Cold Spring HarborLaboratory; the disclosure of which is hereby incorporated by referencein its entirety]. Many commercial kits for carrying out site-specificmutagenesis are well known to those of ordinary skill in the art and arereadily available. Those skilled in the art are credited with theability to select techniques for mutation of a nucleic acid sequence.

Example 33 Oral Bioavailability

Based upon the in vivo data developed, as for example by way ofillustration and not limitation data through the rat model of Example 31supra, oral bioavailability of a modulator of the invention isdetermined. The modulator is administered by oral gavage at dosesranging from 0.1 mg kg⁻¹ to 100 mg kg⁻. Oral administration of themodulator is shown to reduce the level of plasma free fatty acids. Theeffect of the modulator is shown to be dose-dependent and comparable tothe effect after intraperitoneal administration. The dose of modulatorrequired to achieve half-maximal reduction of plasma free fatty acidsthrough oral administration is compared to the dose of modulatorrequired to achieve half-maximal reduction of plasma free fatty acidsthrough intraperitoneal administration. By way of illustration, if saidoral dose is twice said intraperitoneal dose, then the oralbioavailability of the modulator is taken to be 50%. More generally, ifsaid oral dose is θ mg kg⁻¹ and said intraperitoneal dose is ρ mg kg⁻¹,then the oral bioavailability of the modulator as a percentage is takento be [(ρ/θ)×100].

It is readily envisioned that the reference route of administration maybe other than intraperitoneal. In some embodiments, said reference routeof administration is intravenous.

It would be readily apparent to anyone of ordinary skill in the art thatthe aforesaid determination could be modified to utilize a different invivo animal model other than normal Sprague Dawley rats. It would alsobe readily apparent to anyone of ordinary skill in the art that thebioactivity readout in the aforesaid determination could be a parameterother than plasma free fatty acids.

Alternative, physicochemico analytical approaches for assessing oralbioavailability are well known to those of ordinary skill in the art[see, e.g., without limitation: Wong P C et al., Cardiovasc Drug Rev(2002) 20:137-52; and Buchan P et al., Headache (2002) Suppl 2:S54-62;the disclosure of each of which is hereby incorporated by reference inits entirety]. By way of further illustration and not limitation, saidalternative analytical approaches may comprise liquidchromatography-tandem mass spectrometry [Chavez-Eng C M et al., JChromatogrB Analyt Technol Biomed Life Sci (2002) 767:117-29; Jetter Aet al., Clin Pharmacol Ther (2002) 71:21-9; Zimmerman J J et al., J ClinPharmacol (1999) 39:1155-61; and Barrish A et al., Rapid Commun MassSpectrom (1996) 10:1033-7; the disclosure of each of which is herebyincorporated by reference in its entirety].

1. A method of identifying whether a candidate compound is a modulatorof a nicotinic acid GPCR, said receptor comprising an amino acidsequence selected from the group consisting of: (a) SEQ. ID. NO.:36(hRUP25); (b) SEQ. ID. NO.:137 (mRUP25); and (c) SEQ. ID. NO.:139(rRUP25); or an allelic variant, a biologically active mutant, or abiologically active fragment of said amino acid sequence; comprising thesteps of: (a′) contacting the candidate compound with the receptor; and(b′) determining whether the receptor functionality is modulated;wherein a change in receptor functionality is indicative of thecandidate compound being a modulator of a nicotinic acid GPCR.
 2. Amodulator of a nicotinic acid GPCR (RUP25) identified according to themethod of claim 1, provided that the modulator is not identical to acompound selected from the group consisting of:

wherein: R₁ is selected from the group consisting of halogen, hydroxyl,acetylamino, amino, alkoxy, carboalkoxy, alkylthio, monoalkylamino,dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfonyl, saidalkyl groups containing from 1 to 4 carbons, trifluoromethyl,trifluoromethoxy, trifluoromethylthio, methoxymethyl, carboxy, carbamyl,alkanoyloxy containing up to 4 carbon atoms, phenyl, p-chlorophenyl,p-methylphenyl and p-aminophenyl; R₂ is selected from the groupconsisting of halogen, alkannoyloxy containing from 1-4 carbon atoms,carboalkoxy containing from 2 to 5 carbon atoms, carbamyl, N-alkylcarbamyl and N,N-dialkylcarbamyl wherein said alkyl groups contain from1-4 carbon atoms and trifluoromethyl; n is a whole number from 0 to 4;and N-oxides thereof;

R₃ and R₄ are hydrogen, alkyl containing from 1 to 4 carbon atoms orcycloalkyl containing from 3 to 7 carbon atoms; n is a whole number from0 to 4; and N-oxides thereof.

wherein: R₅ and R₆ are each selected from the group consisting of H,halogen, hydroxyl, amino, alkyloxy, alkylthio, monoalkylamino,dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfoxy,alkylsulfony, said alkyl groups containing from 1 to 4 carbons,trifluoromethyl, trifluoromethoxy, trifluoromethylthio, carboxy,carbamyl, alkanoyloxy containing up to 4 carbon atoms, phenyl,p-chlorophenyl, p-methylphenyl and p-aminophenyl; n is a whole numberfrom 0 to 4; and N-oxides thereof;

wherein: at least one of R₇, R₈ and R₉ is C₁₋₆ alkyl and the others arehydrogen atoms; R₁₀ is hydroxy or C₁₋₆ alkoxy, or a salt of thecompounds wherein R₄ is hydoxy with a pharmaceutically acceptable base;

wherein: at least one of R₇, R₈ and R₉ is C₁₋₆ alkyl and the others arehydrogen atoms; each of R₁₁ and R₁₂, which may be the same or different,is hydrogen or C₁₋₆ alkyl;

wherein: at least one of R₁₃ represents an alkyl group of 7-11 carbonatoms and R₁₄ represents H or a lower alkyl group of up to two carbonatoms, and a pharmaceutically acceptable carrier; g)pyrazine-2-carboxylic acid amide, 5-chloro-pyrazine-2-carboxylic acidamide, 5-amino-pyrazine-2-carboxylic acid amide,5-benzyl-pyrazine-2-carboxylic acid amide,6-chloro-pyrazine-2-carboxylic acid amide,6-methoxy-pyrazine-2-carboxylic acid amide,3-chloro-pyrazine-2-carboxylic acid amide, 3-methoxy-pyrazine-2-carboxylic acid amide, pyrazine-2-carboxylic acidethylamide, morpholin-4-yl-pyrazine-2-ylmethanone,5-methyl-pyrazine-2-carboxylic acid (6-methyl-pyrazin-2-yl)-amide,5-methyl-pyrazine-2-carboxylic acid (5-methyl-pyrazin-2-yl)-amide,5-methyl-pyrazine-2-carboxylic acid (3-methyl-pyrazin-2-yl)-amide,(5-methyl-pyrazin-2-yl)-morpholin-4-yl-methanone,5-methyl-pyrazine-2-carboxylic acid hydroxyamide, pyrazine-2-carboxylicacid, 5-amino-pyrazine-2-carboxylic acid, 5-benzyl-pyrazine-2-carboxylicacid, 6-chloro-pyrazine-2-carboxylic acid,6-methoxy-pyrazine-2-carboxylic acid, 3-hydroxy-pyrazine-2-carboxylicacid, 5-methyl-pyrazine-2-carboxylic acid 2-hydroxy-ethyl ester,5-methyl-pyrazine-2-carboxylic acid allyl ester,5-methyl-pyrazine-2-carboxylic acid phenyl ester,5-methyl-pyrazine-2-carboxylic acid ethoxycarbonylmethyl ester,pyrazine-2-carboxylic acid methyl ester or2-methyl-5-(1H-tetrazol-5-yl)-pyrazine; and 4-N-oxides thereof; h)5-(3-(5-Methyl)isoxazolyl)tetrazole; i)5-(5-(3-Methyl)isoxazolyl)tetrazole; j) 5-(3-Quinolyl)tetrazole; k)Nicotinic acid; l) Pyridazine-4-carboxylic acid; m) 3-pyridine aceticacid; n) 5-Methylnicotinic acid; o) 6-Methylnicotinic acid; p) Nicotinicacid-1-oxide; q) 2-Hydroxynicotinic acid; r) Furane-3-carboxylic acid;s) 5-Methylpyrazole-3-carboxylic acid; and t)3-Methylisoxazole-5-carboxylic acid.
 3. A method of modulating theactivity of a nicotinic acid GPCR, said receptor comprising an aminoacid sequence selected from the group consisting of: (a) SEQ. ID. NO.:36(hRUP25); (b) SEQ. ID. NO.:137 (mRUP25); and (c) SEQ. ID. NO.:139(rRUP25); or an allelic variant, a biologically active mutant, or abiologically active fragment of said amino acid sequence; comprising thestep of contacting the receptor with the modulator of claim
 2. 4. Amethod of preventing or treating a disorder of lipid metabolism in anindividual comprising contacting a therapeutically effective amount ofthe modulator of claim 2 with a nicotinic acid GPCR, said receptorcomprising an amino acid sequence selected from the group consisting of:(a) SEQ. ID. NO.:36 (hRUP25); (b) SEQ. ID. NO.:137 (mRUP25); and (c)SEQ. ID. NO.:139 (rRUP25); or an allelic variant or biologically activefragment of said amino acid sequence.
 5. A method of preventing ortreating a metabolic-related disorder in an individual comprisingcontacting a therapeutically effective amount of the modulator of claim2 with a nicotinic acid GPCR, said receptor comprising an amino acidsequence selected from the group consisting of: (a) SEQ. ID. NO.:36(hRUP25); (b) SEQ. ID. NO.:137 (mRUP25); and (c) SEQ. ID. NO.:139(rRUP25); or an allelic variant or biologically active fragment of saidamino acid sequence.
 6. A method of preparing a composition whichcomprises identifying a modulator of a nicotinic acid GPCR and thenadmixing a carrier and the modulator, wherein the modulator isidentifiable by the method of claim 1 and provided that the modulator isnot identical to a compound selected from the group consisting of:

wherein: R₁ is selected from the group consisting of halogen, hydroxyl,acetylamino, amino, alkoxy, carboalkoxy, alkylthio, monoalkylamino,dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfonyl, saidalkyl groups containing from 1 to 4 carbons, trifluoromethyl,trifluoromethoxy, trifluoromethylthio, methoxymethyl, carboxy, carbamyl,alkanoyloxy containing up to 4 carbon atoms, phenyl, p-chlorophenyl,p-methylphenyl and p-aminophenyl; R₂ is selected from the groupconsisting of halogen, alkannoyloxy containing from 1-4 carbon atoms,carboalkoxy containing from 2 to 5 carbon atoms, carbamyl, N-alkylcarbamyl and N,N-dialkylcarbamyl wherein said alkyl groups contain from1-4 carbon atoms and trifluoromethyl; n is a whole number from 0 to 4;and N-oxides thereof;

R₃ and R₄ are hydrogen, alkyl containing from 1 to 4 carbon atoms orcycloalkyl containing from 3 to 7 carbon atoms; n is a whole number from0 to 4; and N-oxides thereof

wherein: R₅ and R₆ are each selected from the group consisting of H,halogen, hydroxyl, amino, alkyloxy, alkylthio, monoalkylamino,dialkylamino, N-alkylcarbamyl, N,N-dialkylcarbamyl, alkylsulfoxy,alkylsulfony, said alkyl groups containing from 1 to 4 carbons,trifluoromethyl, trifluoromethoxy, trifluoromethylthio, carboxy,carbamyl, alkanoyloxy containing up to 4 carbon atoms, phenyl,p-chlorophenyl, p-methylphenyl and p-aminophenyl; n is a whole numberfrom 0 to 4; and N-oxides thereof;

wherein: at least one of R₇, R₈ and R₉ is C₁₋₆ alkyl and the others arehydrogen atoms; R₁₀ is hydroxy or C₁₋₆ alkoxy, or a salt of thecompounds wherein R₄ is hydoxy with a pharmaceutically acceptable base;

wherein: at least one of R₇, R₈ and R₉ is C₁₋₆ alkyl and the others arehydrogen atoms; each of R₁₁ and R₁₂, which may be the same or different,is hydrogen or C₁₋₆ alkyl;

wherein: at least one of R₁₃ represents an alkyl group of 7-11 carbonatoms and R₁₄ represents H or a lower alkyl group of up to two carbonatoms, and a pharmaceutically acceptable carrier; g)pyrazine-2-carboxylic acid amide, 5-chloro-pyrazine-2-carboxylic acidamide, 5-amino-pyrazine-2-carboxylic acid amide,5-benzyl-pyrazine-2-carboxylic acid amide,6-chloro-pyrazine-2-carboxylic acid amide,6-methoxy-pyrazine-2-carboxylic acid amide,3-chloro-pyrazine-2-carboxylic acid amide,3-methoxy-pyrazine-2-carboxylic acid amide, pyrazine-2-carboxylic acidethylamide, morpholin-4-yl-pyrazine-2-ylmethanone,5-methyl-pyrazine-2-carboxylic acid (6-methyl-pyrazin-2-yl)-amide,5-methyl-pyrazine-2-carboxylic acid (5-methyl-pyrazin-2-yl)-amide,5-methyl-pyrazine-2-carboxylic acid (3-methyl-pyrazin-2-yl)-amide,(5-methyl-pyrazin-2-yl)-morpholin-4-yl-methanone,5-methyl-pyrazine-2-carboxylic acid hydroxyamide, pyrazine-2-carboxylicacid, 5-amino-pyrazine-2-carboxylic acid, 5-benzyl-pyrazine-2-carboxylicacid, 6-chloro-pyrazine-2-carboxylic acid,6-methoxy-pyrazine-2-carboxylic acid, 3-hydroxy-pyrazine-2-carboxylicacid, 5-methyl-pyrazine-2-carboxylic acid 2-hydroxy-ethyl ester,5-methyl-pyrazine-2-carboxylic acid allyl ester,5-methyl-pyrazine-2-carboxylic acid phenyl ester,5-methyl-pyrazine-2-carboxylic acid ethoxycarbonylmethyl ester,pyrazine-2-carboxylic acid methyl ester or2-methyl-5-(1H-tetrazol-5-yl)-pyrazine; and 4-N-oxides thereof; h)5-(3-(5-Methyl)isoxazolyl)tetrazole; i)5-(5-(3-Methyl)isoxazolyl)tetrazole; j) 5-(3-Quinolyl)tetrazole; k)Nicotinic acid; l) Pyridazine-4-carboxylic acid; m) 3-pyridine aceticacid; n) 5-Methylnicotinic acid; o) 6-Methylnicotinic acid; p) Nicotinicacid-1-oxide; q) 2-Hydroxynicotinic acid; r) Furane-3-carboxylic acid;s) 5-Methylpyrazole-3-carboxylic acid; and t)3-Methylisoxazole-5-carboxylic acid.
 7. A pharmaceutical orphysiologically acceptable composition comprising, consistingessentially of, or consisting of the modulator of claim
 2. 8. A methodof changing lipid metabolism comprising providing or administering to anindividual in need of said change said pharmaceutical or physiologicallyacceptable composition of claim
 7. 9. A method of preventing or treatinga metabolic-related disorder comprising providing or administering to anindividual in need of said prevention or treatment said pharmaceuticalor physiologically acceptable composition of claim
 7. 10. A method ofusing the modulator of claim 2 for the preparation of a medicament forthe treatment of a disorder in lipid metabolism in an individual.
 11. Amethod of using the modulator of claim 2 for the preparation of amedicament for the treatment of a metabolic-related disorder in anindividual.
 12. A method of identifying whether a candidate compoundbinds to a nicotinic acid GPCR, said receptor comprising an amino acidsequence selected from the group consisting of: (a) SEQ. ID. NO.:36(hRUP25); (b) SEQ. ID. NO.:137 (mRUP25); and (c) SEQ. ID. NO.:139(rRUP25); or an allelic variant or a biologically active fragment ofsaid amino acid sequence; comprising the steps of: (a′) contacting thereceptor with a labeled reference compound known to bind to the GPCR inthe presence or absence of the candidate compound; and (b′) determiningwhether the binding of said labeled reference compound to the receptoris inhibited in the presence of the candidate compound; wherein saidinhibition is indicative of the candidate compound binding to anicotinic acid GPCR.
 13. A method of making a mouse geneticallypredisposed to a metabolic-related disorder or a disorder of lipidmetabolism comprising the step of knocking out the gene encoding thepolypeptide of SEQ. ID. NO.:137 (mRUP25) or the polypeptide of SEQ. ID.NO.:151 (mRUP19).
 14. A knockout mouse according to the method of claim13.
 15. A method of using the knockout mouse of claim 14 to identifywhether a candidate compound has therapeutic efficacy for the preventionor treatment of said metabolic-related disorder or said disorder oflipid metabolism.
 16. A method of making a rat genetically predisposedto a metabolic-related disorder or a disorder of lipid metabolismcomprising the step of knocking out the gene encoding the polypeptide ofSEQ. ID. NO.:139 (rRUP25) or the polypeptide of SEQ. ID. NO.:157(rRUP19).
 17. A knockout rat according to the method of claim
 16. 18. Amethod of using the knockout rat of claim 17 to identify whether acandidate compound has therapeutic efficacy for the prevention ortreatment of said metabolic-related disorder or said disorder of lipidmetabolism.
 19. An isolated EFA-hRUP25 polynucleotide selected from thegroup consisting of: (a) a polynucleotide comprising the nucleotidesequence of SEQ. ID. NO.:158; (b) a polynucleotide having the nucleotidesequence of SEQ. ID. NO.:158; (c) a polynucleotide comprising apolynucleotide encoding the polypeptide having the amino acid sequenceof SEQ. ID. NO.:159 or a biologically active fragment of saidpolypeptide; and (d) a polynucleotide encoding the polypeptide havingthe amino acid sequence of SEQ. ID. NO.:159 or a biologically activefragment of said polypeptide.
 20. An isolated EFA-hRUP25 polypeptideselected from the group consisting of: (a) a polypeptide comprising theamino acid sequence of SEQ. ID. NO.:159, or a biologically activefragment of said polypeptide; and (b) a polypeptide having the aminoacid sequence of SEQ. ID. NO.:159, or a biologically active fragment ofsaid polypeptide.
 21. A recombinant vector comprising the polynucleotideof claim
 19. 22. A host cell comprising the recombinant vector of claim21.